Apparatus and methods for conducting assays and high throughput screening

ABSTRACT

The present invention provides microfluidic devices and methods for using the same. In particular, microfluidic devices of the present invention are useful in conducting a variety of assays and high throughput screening. Microfluidic devices of the present invention include elastomeric components and comprise a main flow channel; a plurality of branch flow channels; a plurality of control channels; and a plurality of valves. Preferably, each of the valves comprises one of the control channels and an elastomeric segment that is deflectable into or retractable from the main or branch flow channel upon which the valve operates in response to an actuation force applied to the control channel.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/416,418, filed Oct. 23, 2003, which is a U.S. national phaseapplication of International Patent Application No. PCT/US2001/044869,filed Nov. 16, 2001, which claims priority benefit of U.S. ProvisionalPatent Application No. 60/249,327, filed Nov. 16, 2000, and U.S.Provisional Patent Application No. 60/281,946, filed Apr. 6, 2001, andU.S. Provisional Patent Application No. 60/281,948, filed Apr. 6, 2001,all of which are incorporated herein by reference in their entirety

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of Grant No.HG-01642-02 awarded by the National Institutes of Health.

FIELD OF THE INVENTION

The present invention relates to microfluidic apparatus and methods forconducting a variety of assays and high throughput screening.

BACKGROUND OF THE INVENTION

There are several goals in the development of biological assays,including utilization of a minimal amount of assay components andsample, simplicity in operation and high throughput capability. Assayspreferably require a minimal amount of assay components in order tominimize costs; this becomes a particular issue if certain assaycomponents are expensive and/or a large number of assays are to beconducted. Ideally, assays require only a minimal amount of samplebecause often only a very limited amount of sample is available. Thegoal of simplicity of operation often means that the assay is preferablyconducted in an integrated format in which all or most aspects of theassay can be conducted with a single device and minimal instrumentation.The goal of high throughput has become increasingly important in view ofthe trend in current research and drug discovery efforts to screen hugelibraries of compounds to identify those that have a desired activity.

To address some of these problems, particularly the issue of minimizingthe amount of sample and assay agents required to conduct an analysis,considerable effort has been invested in the development of microfluidicdevices to conduct assays. These devices are characterized by usingminute channels for the introduction, transport and mixing of thesamples and agents necessary to conduct an assay. Unfortunately, currentmicrofluidic devices suffer from a number of shortcomings that limittheir usefulness. For example, current microfluidic devices often aremanufactured from silicon chips with channels being etched intodifferent silicon layers using established semi-conductor technologies.Such chips, however, are brittle and the stiffness of the material oftennecessitates high actuation forces. These forces and stresses can causelayers in a multilayer chip to separate from one another. The stiffnessof the devices also imposes significant constraints on options forcontrolling solution flow through the microchannels.

Furthermore, solution flow is controlled at least in part through theuse of electrodes to generate electric fields to move molecules andsolution via electrophoresis and/or electroosmosis. Reliance onelectrodes, however, creates several problems. One problem is that gasis often generated at the electrodes. This can increase pressure withinthe device potentially causing separation of microfabricated layers. Theincreased pressure and gas bubbles can also interfere with solution flowthrough the channels. Additionally, often an elaborate network ofelectrodes is required in order to achieve the desired level of controlover solution transport. Fabrication of such a network can becomplicated and increases the expense of the devices. The need for suchnetworks also becomes particularly problematic if a device is to beprepared that includes a large number of branch channels to facilitatemultiplexed and high throughput assay capabilities. Moreover, the use ofelectrical fields to control solution flow can be problematic forapplications involving cells as application of the electric fields cannegatively affect the cells, often killing them. Consequently, thereremains a significant need for improved microfluidic devices,particularly those that are amendable to a wide range of cellular assaysand that have high throughput capabilities.

BRIEF SUMMARY OF THE INVENTION

A variety of microfluidic devices and methods for conducting assays andsyntheses are provided herein. Unlike conventional microfluidic devices,the devices disclosed herein include elastomeric components. In someinstances, the entire device is manufactured from elastomeric materials.The devices can be utilized in a wide variety of applications, includingcell assays and high throughput screening applications, as well as inthe synthesis of a variety of compounds.

One aspect of the present invention provides a microfluidic devicecomprising: (a) a main flow channel adapted to allow the flow of asolution therethrough; (b) a plurality of branch flow channels, whereineach branch flow channel is in fluid communication with the main flowchannel and comprises a detection region; (c) a plurality of controlchannels; and (d) a plurality of valves operatively disposed withrespect to the main flow channel and/or the plurality of branch flowchannels to regulate flow of the solution through the main and branchflow channels. Each of the valves comprises one of the control channelsand an elastomeric segment that is deflectable into or retractable fromthe main or branch flow channel upon which the valve operates inresponse to an actuation force applied to the control channel. When theelastomeric segment is positioned in the flow channel, it restricts theflow of solution therethrough.

In one embodiment, the control channel of each valve is separated fromthe flow channel in which the valve operates by the elastomeric segment.

In another embodiment, the elastomeric segment is deflectable into thebranch flow channel. While in another embodiment, the elastomericsegment is retractable from the branch flow channel.

Still in another embodiment, the microfluidic device further comprises asolution inlet in fluid communication with the main flow channel forintroduction of the solution. In addition, the microfluidic device canfurther comprise a second solution inlet for each branch flow channel influid communication therewith for introduction of a second solution.Furthermore or alternatively, the microfluidic device can comprise anauxiliary inlet in fluid communication with the main flow channel. Inaddition, it can also comprise an inlet flow channel in fluidcommunication with the main flow channel. Preferably, the solution inletand the auxiliary inlet are in fluid communication with the inlet flowchannel.

Yet in another embodiment, the microfluidic device comprises a detectoroperatively disposed with respect to at least one of the detectionsections to detect an event or agent within the detection region. Thedetector can be a device which detects an optical signal from thedetection region. In one particular embodiment, the detector can detecta fluorescence emission, fluorescence polarization or fluorescenceresonance energy transfer. Preferably, the detector can performtime-resolved fluorescence measurements or fluorescence correlationspectroscopy. In another embodiment, the detection is an opticalmicroscope, a confocal microscope or a laser scanning confocalmicroscope. Still in another particular embodiment, the detector is anon-optical sensor selected from the group consisting of a temperaturesensor, a conductivity sensor, a potentiometric sensor and anamperometric sensor.

Still in another embodiment, each branch flow channel is adapted toallow the flow of a second solution therethrough. In this manner, thesolution, which flows through the main fluid channel can contain assaycomponents for an assay that is conducted in each of the differentbranch flow channels and the second solution can contain a test agent.

In another embodiment, the main flow channel is one of a plurality ofmain flow channels, each adapted to allow the flow of a first solutiontherethrough and in fluid communication with each of the branch flowchannels.

In yet another embodiment, each of the branch flow channels intersectwith the main flow channel at a different intersection, and furthercomprising a chamber located at each of the intersections, the chamberbeing adapted to collect solution therein.

Yet still in another embodiment, the microfluidic device furthercomprises a plurality of pumps. Preferably, each pump is operativelydisposed with respect to one of the branch flow channels such thatsolution flow through each of the branch flow channels can be regulatedby one of the pumps. In one particular embodiment, each of the pluralityof pumps comprises at least three control channels each formed within anelastomeric material and separated from the branch flow channel by asection of an elastomeric membrane, the membrane being deflectable intothe branch flow channel in response to an actuation force.

Still in another embodiment, the microfluidic device further comprises aplurality of mixers. Each mixer is operatively disposed with respect toone of the branch flow channels and is adapted to receive and mixdifferent solutions flowing through one of the branch flow channels.Furthermore, an operatively disposed temperature controller can bepresent to regulate temperature within the mixer. Preferably, the mixercomprises

(i) an inlet section and an outlet section

(ii) a looped flow channel in fluid communication with the inlet andoutlet section; and

(iii) two sets of control channels, each set comprising at least threecontrol channels, and each control channel separated from the loopedflow channel by an elastomeric membrane that is deflectable into thelooped flow channel in response to an actuation force, and wherein themixer is inserted into one of the branch flow channels such thatsolution from the branch flow channel can enter via the inlet sectionand reenter the branch flow channel via the outlet section, and solutionwithin the mixer can be cycled through the looped flow channel byactuating the control channels with the actuation force which is appliedto the control channels within a set in a repeating sequence. Moreoverthe detection section can also comprise the mixer.

In another embodiment, the microfluidic device further comprises aplurality of temperature controllers. Each temperature controller isoperatively disposed with respect to one of the branch channels. Suchtemperature controller can be operative disposed within the microfluidicdevice to regulate temperature within the detection section.

Still in another embodiment, the microfluidic device further comprises aplurality of separation units, where each separation unit is operativelydisposed with respect to one of the branch flow channels. Preferably,each separation unit comprises a semi-permeable membrane that separatesone of the branch flow channels and a collection channel. The membraneallows passage of certain agents in a solution flowing through thebranch flow channel to pass through the semi-permeable membrane into thecollection flow channel. Alternatively, the separation unit comprises aseparation material that separates molecules on the basis of affinity,size, charge or mobility.

Yet in another embodiment, the microfluidic device further comprises (e)an inlet in fluid communication with the main flow channel forintroduction of the first solution and an inlet for each branch flowchannel in fluid communication therewith for introduction of the secondsolution, the first solution containing assay components for an assaythat is conducted in each of the different branch flow channels and thesecond solution containing a test agent; (f) a plurality of chamberspositioned at points where the main flow channel and the branch flowchannels intersect, the chambers being adapted to collect solutiontherein; (g) a plurality of pumps, each pump being operatively disposedwith respect to one of the branch flow channels such that solution flowthrough each of the branch flow channels can be regulated by one of thepumps; and (h) a plurality of mixers, each mixer being operativelydisposed with respect to one of the branch flow channels and beingadapted to receive and mix different solutions flowing through one ofthe branch flow channels.

In yet another embodiment, the microfluidic device further comprises apair of valves operatively disposed with respect to each of the branchflow channels. The pair of valves are preferably disposed with respectto one another such that when the elastomeric segment of each valve ofthe pair extends into the branch flow channel a holding space is formedbetween the segments in which the solution can be retained. In addition,the microfluidic device can further comprises a plurality of controlvalves operatively disposed with respect to the main flow channel.Preferably, the control valves are positioned along the main flowchannel such that by selectively actuating the control valves solutioncan be controllably introduced into the branch flow channels. Moreover,the control valves each comprise one of the control channels and anelastomeric segment that is deflectable into or retractable from themain flow channel in response to an actuation force applied to thecontrol channel. Furthermore, the elastomeric segments of at least onepair of valves each comprise one or more protrusions. The protrusionsare adapted to allow for the solution to flow through the holding spaceonce the elastomeric segments are deflected into the branch flow channelwhile retaining a particle of a predetermined size that is present inthe solution within the holding space of the at least one pair ofvalves. The particle can be any non-soluble material including cells.

Preferably, the branch flow channel is present within an elastomericmaterial. Moreover, a segment of the branch flow channel opposite theelastomeric segment of each valve of at least one pair of valvescomprises one or more elastomeric protrusions. The protrusions adaptedto allow for solution to flow through the holding space once theelastomeric segment is deflected into the branch flow channel whileretaining a particle of a predetermined size that is present in thesolution within the holding space of the at least one pair of valves.

In another embodiment, the branch flow channels are formed within anelastomeric material; the elastomeric segments of at least one pair ofvalves comprise one or more protrusions; a segment of the branch flowchannel opposite the elastomeric segments of the at least one valve paircomprises one or more protrusions, and wherein the protrusions of theelastomeric segment and the protrusions of the branch flow channel areadapted to allow for solution to flow through the holding space of theat least one valve pair once the elastomeric segments are deflected intothe branch flow channel while retaining a particle of a predeterminedsize that is present in the solution within the holding space. Still inanother embodiment, the microfluidic device further comprises aplurality of chambers positioned at points where an inlet flow channeland the branch flow channels intersect, the chambers adapted for storingsolution.

Yet in another embodiment, the microfluidic device further comprises aplurality of branch control valves, wherein a branch control valve isoperatively disposed with respect to each of the branch flow channelsand each branch control valve comprises an elastomeric segment thatseparates one of the branch flow channels and one of the controlchannels and that is deflectable into or retractable from the branchflow channel upon which the valve operates in response to an actuationforce applied to the control channel.

In one embodiment, each branch flow channel is in communication with apump. Preferably, the pump comprises at least three control channels,each formed within an elastomeric material and separated from one of thebranch flow channels by an elastomeric membrane, the membrane beingdeflectable into the branch flow channel in response to an actuationforce.

In another embodiment, each branch flow channel comprises a particleenrichment section that selectively retains particles of interest.Preferably, the enrichment section contains a ligand that specificallybinds to particle (s) that are present in the solution. In addition, thedetection region, preferably, includes the holding space and a detectoris disposed to detect particle (s) within the holding spaces within theplurality of branch channels.

In one particular embodiment, the detection region includes the holdspace.

In another embodiment, the detection region is located at a section ofthe branch flow channel other than the holding space.

Another aspect of the present invention provides a microfluidic devicefor conducting cellular assays, comprising (a) a main flow channel; (b)an input adapted to receive a first solution and in fluid communicationwith the main flow channel, whereby solution introduced into the inputcan flow into the main flow channel; (d) a plurality of controlchannels; (e) a plurality of chambers positioned along the main flowchannel and in fluid communication therewith, such that solution flowingthrough the main flow channel can be stored in the chambers; (f) aplurality of branch flow channels, wherein each branch flow channel isin fluid communication with a branch inlet, each branch inlet adapted toreceive a second solution and in fluid communication with one of thechambers, different branch flow channels being in fluid communicationwith different chambers; (g) a plurality of storage valves, wherein apair of storage valves are operatively disposed with respect to each ofthe branch flow channels, and wherein each of the storage valvescomprises an elastomeric segment that separates one of the branch flowchannels and one of the control channels and that is deflectable into orretractable from the flow channel upon which the valve operates inresponse to an actuation force applied to the control channel, thestorage valves of a pair being disposed with respect to one another suchthat when the elastomeric segment of each storage valve of the pairextends into the branch flow channel a holding space is formed betweenthe segments in which one or more cells can be retained; and (h) one ormore pumps to transport the first and/or second solution through thebranch flow channels. Preferably, each pump comprises at least threecontrol channels, each formed within an elastomeric material andseparated from one of the branch flow channels by a section of anelastomeric membrane, the membrane being deflectable into the branchflow channel in response to an actuation force.

Still another aspect of the present invention provides a method ofconducting a cellular analysis, comprising

(a) providing a microfluidic device comprising

-   -   (i) a flow channel comprising a detection region;    -   (ii) a plurality of control channels; and    -   (iii) a pair of storage valves operatively disposed with respect        to the flow channel, wherein each storage valve comprises one of        the control channels, which control channel is separated from        the flow channel by an elastomeric membrane, the elastomeric        membrane being deflectable into the flow channel in response to        an actuation force applied to the control channel, the two        control channels of the storage valves being disposed relative        to one another such that deflection of the elastomeric membrane        of the respective control channels into the flow channel forms a        holding space within the flow channel;

(b) introducing a sample containing one or more cells into the flowchannel;

(c) actuating the storage valves to hold at least one of the cellswithin the holding space; and

(d) performing an assay by contacting the cells within the holding spacewith a solution containing one or more assay agents.

Preferably, the elastomeric membranes of the storage valves eachcomprise one or more protrusions. The protrusions are adapted to allowfor solution to flow through the holding space once the elastomericmembranes are deflected into the flow channel while retaining the cell(s) within the holding space.

In one embodiment, the flow channel is formed within an elastomericmaterial and a segment of the flow channel opposite the elastomericmembrane of the storage valves comprises one or more elastomericprotrusions, the protrusions adapted to allow for solution to flowthrough the holding space once the elastomeric membranes are deflectedinto the flow channel while retaining the cell (s) within the holdingspace.

In another embodiment, the flow channel is formed within an elastomericmaterial; the elastomeric segments of the storage valves comprise one ormore protrusions; a segment of the flow channel opposite the elastomericmembrane of each storage valve comprises one or more protrusions, andwherein the protrusions of the elastomeric membrane and the protrusionsof the branch flow channel are adapted to allow for solution to flowthrough the holding space once the elastomeric membranes are deflectedinto the branch flow channel while retaining the cell (s) within theholding space.

Still in another embodiment, the microfluidic device further comprises achamber adapted for storing solution in fluid communication with theflow channel and the method further comprises transporting solutionand/or cells back and forth between the chamber and the holding space.

Preferably, the flow channel is in communication with a pump. And thesample is transported through the flow channel under the action of thepump. Preferably, the pump comprises at least three of the controlchannels, each formed within an elastomeric material and separated fromthe flow channel by an elastomeric membrane, each membrane beingdeflectable into the flow channel in response to an actuation force,whereby sample is transported along the flow channel.

In one embodiment, the flow channel comprises a cell enrichment sectionthat selectively retains cells of interest, and the method furthercomprises transporting the sample through the enrichment section.Preferably, the enrichment section contains a ligand that specificallybinds to a receptor on target cells.

In one embodiment of the present invention, the assaying step comprisesdetecting an event and/or agent within the detection section with adetector.

In another embodiment, the assay step comprises flowing a solutionthrough the holding spaces after the storage valves have been actuated.

In one particular embodiment, the solution flowed through the holdingspaces is culture medium for the cells within the holding space.

In another embodiment, the solution comprises a cellular stain.

Still in another embodiment, the assay comprises detecting cellularmorphology.

Yet in another embodiment, the assay is a cell reporter assay.

In another embodiment, the assay comprises detecting binding between aligand and a cell receptor.

In another embodiment, the assay comprises measuring cell membranepotential.

Still in another embodiment, the assay is an assay for detecting a toxiceffect on cells or a cell death assay.

Yet in another embodiment, the assay is a cell proliferation assay, anassay for dysfunction of mitochondrial membrane potential, caspaseactivation, or cytochrome c release from cells.

In still yet another embodiment, the assay is a cell lysis assay.

In another embodiment, the assay is an antimicrobial assay. Anotheraspect of the present invention provides a method of conducting acellular analysis, comprising

(a) providing a microfluidic device comprising

-   -   (i) a main flow channel;    -   (ii) a plurality of branch flow channels, each branch flow        channel being in fluid communication with the main flow channel;    -   (iii) a plurality of control channels; and    -   (iv) a plurality of storage valves, wherein a pair of storage        valves are operatively disposed with respect to each of the        branch flow channels, and wherein each of the storage valves        comprises an elastomeric segment that separates one of the        branch flow channels and one of the control channels and that is        deflectable into or retractable from the flow channel upon which        the valve operates in response to an actuation force applied to        the control channel, the storage valves of a pair being disposed        with respect to one another such that when the elastomeric        segment of each storage valve of the pair extends into the        branch flow channel a holding space is formed between the        segments in which one or more cells can be retained;

(b) introducing a solution containing one or more cells into each of thebranch flow channels;

(c) actuating the storage valves within each of the branch flow channelsto form the holding space to hold at least one of the cells within thesample introduced into the branch flow channel; and

(d) performing an assay by contacting the cells within the holding spacewith a solution containing one or more assay agents.

In one particular embodiment, different cells are introduced intodifferent branch flow channels and the assaying step comprisescontacting the cells within each branch flow channel with the same assaysolution.

In another embodiment, cells of the same type are introduced into eachof the flow channels and the assaying step comprises contacting thecells within different branch flow channels with different assaysolutions.

Yet another aspect of the present invention provides a method forconducting an assay comprising, comprising:

(a) providing a microfluidic device comprising

-   -   (i) a main flow channel;    -   (ii) a plurality of branch flow channels, each in fluid        communication with the main flow channel and comprising a        detection section;    -   (iii) a plurality of control channels; and    -   (iv) a plurality of valves operatively disposed with respect to        the main flow channel and/or the plurality of branch flow        channels to regulate flow of the first and second solutions        therethrough, wherein each of the valves comprises one of the        control channels and an elastomeric segment that is deflectable        into or retractable from the main or branch flow channel upon        which the valve operates in response to an actuation force        applied to the control channel, the elastomeric segment when        positioned in the flow channel restricting solution flow        therethrough;

(b) introducing a first assay solution into the main flow channel and asecond assay solution into each of the plurality of branch flowchannels;

(c) actuating one or more of the valves to control flow of the first andsecond assay solutions through the main and branch flow channels,whereby the first and second assay solutions are brought into contactwith one another to form an assay solution mixture; and

(d) assaying each of the assay solution mixtures for a desired propertywithin the detection section.

In one embodiment, the control channel of each valve is separated fromthe flow channel in which the valve operates by the elastomeric segment.In particular, the method is a method for screening a plurality oftarget agents for the desired property; the first assay solution is asolution comprising one or more assay agents and the second assaysolution is a solution comprising one of the test compounds; theintroducing step comprises introducing a different test compound intoeach of the plurality of branch flow channels; and the assaying stepcomprises identifying at least one test compound having the desiredactivity.

In another embodiment, the microfluidic device further comprises a firstinlet in fluid communication with the main flow channel and a secondinlet for each branch flow channel, and wherein the first assay solutionis introduced via the first inlet and the second assay solutionintroduced via the second inlet.

Yet in another embodiment, the microfluidic device further comprises aplurality of pumps, each pump being operatively disposed with respect toone of the branch flow channels, and wherein the method furthercomprises actuating the pump of each branch channel to transport thefirst and/or second assay solution through the branch flow channel.Preferably, each of the plurality of pumps comprises a at least threecontrol channels each formed within an elastomeric material andseparated from one of the branch flow channels by a section of anelastomeric membrane, the membrane being deflectable into the branchflow channel from which it is separated in response to an actuationforce.

Still in another embodiment, the microfluidic device further comprises aplurality of mixers, each mixer being operatively disposed with respectto one of the branch flow channels, and wherein the method furthercomprises mixing the first assay solution and the second assay solutionwith the mixer to form the assay solution mixture prior to the assayingstep. Preferably, the mixer comprises (i) an inlet section and an outletsection (ii) a looped flow channel in fluid communication with the inletand outlet section; and (iii) two sets of control channels, each setcomprising at least three control channels, and each control channelseparated from the looped flow channel by an elastomeric membrane thatis deflectable into the looped flow channel in response to an actuationforce, and wherein the mixing step comprises transporting the first andsecond assay solution into the mixer via the inlet section and mixingthe first and second solution in the looped flow channel by applying theactuation force to the control channels within a set as part of arepeating sequence.

In still another embodiment, the microfluidic device further comprises aplurality of temperature controllers, each temperature controller beingoperatively disposed with respect to one of the branch channels, and themethod further comprises actuating the temperature controller toregulate the temperature of the first assay solution, the second assaysolution and/or the assay solution mixture. Preferably, the temperaturecontroller is disposed to regulate the temperature of the assay solutionmixture within the detection section.

Yet in another embodiment, the microfluidic device further comprises aplurality of mixers, each mixer being operatively disposed with respectto one of the branch flow channels, and wherein the method furthercomprises mixing the first assay solution and the second assay solutionwith the mixer to form the assay solution mixture prior to the assayingstep and wherein the temperature controller is disposed to regulate thetemperature of the assay solution mixture within the mixer.

Still in another embodiment, the microfluidic device further comprises aplurality of separation units, each separation unit being positionedwithin one of the branch flow channels, and wherein the method furthercomprises (e) transporting a mixture of the first and second assaysolutions through the separation unit whereby one or more unwantedcompounds are removed to form the assay solution mixture; and (f)transporting the assay solution mixture to the detection section.

Yet still in another embodiment, the microfluidic device furthercomprises a plurality of mixers and a plurality of separation units,each mixer being operatively disposed with respect to one of the branchflow channels, and each separation unit being positioned within one ofthe branch flow channels, and wherein the method further comprises (e)mixing the first assay solution and the second assay solution with themixer; (f) transporting the mixture of the first and second assaysolutions through the separation unit whereby one or more unwantedcompounds are removed to form the assay solution mixture; and (g)transporting the assay solution mixture to the detection section.Preferably, each separation unit comprises a semi-permeable membranethat separates one of the branch flow channels and a collection channel,the membrane allowing passage of certain agents in the first or secondassay solution or a mixture thereof flowing through the branch flowchannel to pass through the membrane and into the collection flowchannel. Alternatively, the separation unit comprises a separationmaterial that separates molecules on the basis of affinity, size, chargeor mobility.

Still in another embodiment, the microfluidic device further comprises adetector operatively disposed with respect to at least one of thedetection sections, and the assaying step comprises detecting an eventor agent within the detection section.

Yet in another embodiment, the desired property is the ability of the atleast one test agent to promote or inhibit binding between a ligand andan antiligand. In one particular embodiment, the antiligand is a proteinreceptor and the ligand is the receptor's cognate ligand. In anotherparticular embodiment, the ligand is an antigen and the antiligand is anantibody that specifically binds to the antigen. Still in anotherparticular embodiment, the antiligand is an enzyme and the ligand is anenzyme substrate or inhibitor of the enzyme. Yet still in anotherparticular embodiment, the ligand is a nucleic acid and the antiligandis a nucleic acid binding protein.

Still in another embodiment, the desired property is the ability of theat least one test agent to trigger a signal transduction pathway.

Still yet in another embodiment, the desired property is the ability ofthe at least one test agent to inhibit microbial growth.

In another embodiment, the desired property is the ability of the atleast one test agent to trigger cell death.

Yet in another embodiment, the desired property is the ability of the atleast one test agent to trigger apoptosis in a cell.

Still in another embodiment, the assay comprises assaying for inhibitionof cell proliferation, assaying for dysfunction of mitochondrialmembrane function, assaying for caspase activation or assaying forcytochrome c release from a cell.

Yet in another embodiment, the assaying step comprises conducting anassay in a homogenous format.

In another embodiment, the assaying step comprises conducting an assayin a heterogenous format.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs.

The following references provide one of skill with a general definitionof many of the terms used in this invention: Singleton et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed. 1994); THECAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); THEGLOSSARY OF GENETICS, 5TH ED., R. Rieger et al. (eds.), Springer Verlag(1991); and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY(1991). As used herein, the following terms have the meanings ascribedto them unless specified otherwise.

The term “elastomer” and “elastomeric” has its general meaning as usedin the art. Thus, for example, Allcock et al. (Contemporary PolymerChemistry, 2nd Ed.) describes elastomers in general as polymers existingat a temperature between their glass transition temperature andliquefaction temperature. Elastomeric materials exhibit elasticproperties because the polymer chains readily undergo torsional motionto permit uncoiling of the backbone chains in response to a force, withthe backbone chains recoiling to assume the prior shape in the absenceof the force. In general, elastomers deform when force is applied, butthen return to their original shape when the force is removed. Theelasticity exhibited by elastomeric materials can be characterized by aYoung's modulus. The elastomeric materials utilized in the microfluidicdevices disclosed herein typically have a Young's modulus of betweenabout 1 Pa-1 TPa, in other instances between about 10 Pa-100 GPa, instill other instances between about 20 Pa-1 GPa, in yet other instancesbetween about 50 Pa-10 MPa, and in certain instances between about 100Pa-1 MPa. Elastomeric materials having a Young's modulus outside ofthese ranges can also be utilized depending upon the needs of aparticular application.

Some of the microfluidic devices described herein are fabricated from anelastomeric polymer such as GE RTV 615 (formulation), a vinyl-silanecrosslinked (type) silicone elastomer (family). However, the presentmicrofluidic systems are not limited to this one formulation, type oreven this family of polymer; rather, nearly any elastomeric polymer issuitable. Given the tremendous diversity of polymer chemistries,precursors, synthetic methods, reaction conditions, and potentialadditives, there are a large number of possible elastomer systems thatcan be used to make monolithic elastomeric microvalves and pumps. Thechoice of materials typically depends upon the particular materialproperties (e.g., solvent resistance, stiffness, gas permeability,and/or temperature stability) required for the application beingconducted. Additional details regarding the type of elastomericmaterials that can be used in the manufacture of the components of themicrofluidic devices disclosed herein are set forth in U.S. applicationSer. No. 09/605,520, filed Jun. 27, 2000, U.S. application Ser. No.09/724,784, filed Nov. 28, 2000, and PCT publication WO 01/01025, eachof which are incorporated herein by reference in their entirety.

A “ligand” generally refers to any molecule that binds to an antiligandto form a ligand/antiligand pair. Thus, a ligand is any molecule forwhich there exists another molecule (i.e., the antiligand) thatspecifically or non-specifically binds to the ligand, owing torecognition of some portion or feature of the ligand.

An “antiligand” is a molecule that specifically or nonspecificallyinteracts with another molecule (i.e., the ligand).

As used herein, the term “binding pair” or “binding partners” refers tofirst and second molecules that specifically bind to each other such asa ligand and an antiligand. In general, “specific binding” of the firstmember of the binding pair to the second member of the binding pair in asample is evidenced by the binding of the first member to the secondmember, or vice versa, with greater affinity and specificity than toother components in the sample. The binding between the members of thebinding pair is typically noncovalent. Binding partners need not belimited to pairs of single molecules. For example, a single ligand canbe bound by the coordinated action of two or more antiligands. Bindingbetween binding pairs or binding partners results in the formation of abinding complex, sometimes referred to as a ligand/antiligand complex orsimply as ligand/antiligand. Exemplary binding pairs include, but arenot limited to: (a) a haptenic or antigenic compound in combination witha corresponding antibody or binding portion or fragment thereof, (b)nonimmunological binding pairs (e.g., biotin-avidin,biotin-streptavidin, biotin-Neutravidin); (c) hormone-hormone bindingprotein; (d) receptor-receptor agonist or antagonist; (e)lectin-carbohydrate; (f) enzyme-enzyme cofactor; (g) enzyme-enzymeinhibitor; (h) and complementary polynucleotide pairs capable of formingnucleic acid duplexes.

An “analyte” refers to the species whose presence, absence and/orconcentration is being detected or assayed.

“Polypeptide,” “peptides” and “protein” are used interchangeably hereinand include a molecular chain of amino acids linked through peptidebonds. The terms do not refer to a specific length of the product. Theterms include post-translational modifications of the polypeptide, forexample, glycosylations, acetylations, phosphorylations and the like,and also can include polypeptides that include amino acid analogs andmodified peptide backbones.

The term “antibody” as used herein includes antibodies obtained fromboth polyclonal and monoclonal preparations, as well as the following:(i) hybrid (chimeric) antibody molecules (see, for example, Winter etal. (1991) Nature 349:293-299; and U.S. Pat. No. 4,816,567); (ii) F(ab′) 2 and F (ab) fragments; (iii) Fv molecules (noncovalentheterodimers, see, for example, Inbar et al. (1972) Proc. Natl. Acad.Sci. USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096);(iv) single-chain Fv molecules (sFv) (see, for example, Huston et al.(1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); (v) dimeric andtrimeric antibody fragment constructs; (vi) humanized antibody molecules(see, for example, Riechmann et al. (1988) Nature 332:323-327; Verhoeyanet al. (1988) Science 239: 1534-1536; and U. K. Patent Publication No.GB 2,276,169, published 21 Sep. 1994); (vii) Mini-antibodies orminibodies (i.e., sFv polypeptide chains that include oligomerizationdomains at their C-termini, separated from the sFv by a hinge region;see, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992)J. Immunology 149B:120-126); and, (vii) any functional fragmentsobtained from such molecules, wherein such fragments retainspecific-binding properties of the parent antibody molecule.

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused herein to include a polymeric form of nucleotides of any length,including, but not limited to, ribonucleotides or deoxyribonucleotides.There is no intended distinction in length between these terms. Further,these terms refer only to the primary structure of the molecule. Thus,in certain embodiments these terms can include triple-, double- andsingle-stranded DNA, as well as triple-, double- and single-strandedRNA. They also include modifications, such as by methylation and/or bycapping, and modified forms of the polynucleotide. More particularly,the terms “nucleic acid,” “polynucleotide,” and “oligonucleotide,”include polydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), any other type ofpolynucleotide which is an N- or C-glycoside of a purine or pyrimidinebase, and other polymers containing nonnucleotidic backbones, forexample, polyamide (e.g., peptide nucleic acids (PNAs)) andpolymorpholino (commercially available from the Anti-Virals, Inc.,Corvallis, Oreg., as Neugene) polymers, and other syntheticsequence-specific nucleic acid polymers providing that the polymerscontain nucleobases in a configuration which allows for base pairing andbase stacking, such as is found in DNA and RNA.

A “probe” is an nucleic acid capable of binding to a target nucleic acidof complementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation, thus forming a duplex structure. The probe binds orhybridizes to a “probe binding site.” The probe can be labeled with adetectable label to permit facile detection of the probe, particularlyonce the probe has hybridized to its complementary target. The labelattached to the probe can include any of a variety of different labelsknown in the art that can be detected by chemical or physical means, forexample. Suitable labels that can be attached to probes include, but arenot limited to, radioisotopes, fluorophores, chromophores, mass labels,electron dense particles, magnetic particles, spin labels, moleculesthat emit chemiluminescence, electrochemically active molecules,enzymes, cofactors, and enzyme substrates. Probes can vary significantlyin size. Some probes are relatively short. Generally, probes are atleast 7 to 15 nucleotides in length. Other probes are at least 20, 30 or40 nucleotides long. Still other probes are somewhat longer, being atleast 50, 60, 70, 80, 90 nucleotides long. Yet other probes are longerstill, and are at least 100, 150, 200 or more nucleotides long. Probescan be of any specific length that falls within the foregoing ranges aswell.

A “primer” is a single-stranded polynucleotide capable of acting as apoint of initiation of template-directed DNA synthesis under appropriateconditions (i.e., in the presence of four different nucleosidetriphosphates and an agent for polymerization, such as, DNA or RNApolymerase or reverse transcriptase) in an appropriate buffer and at asuitable temperature. The appropriate length of a primer depends on theintended use of the primer but typically is at least 7 nucleotides longand, more typically range from 10 to 30 nucleotides in length. Otherprimers can be somewhat longer such as 30 to 50 nucleotides long. Shortprimer molecules generally require cooler temperatures to formsufficiently stable hybrid complexes with the template. A primer neednot reflect the exact sequence of the template but must be sufficientlycomplementary to hybridize with a template. The term “primer site” or“primer binding site” refers to the segment of the target DNA to which aprimer hybridizes. The term “primer pair” means a set of primersincluding a 5′ “upstream primer” that hybridizes with the complement ofthe 5′ end of the DNA sequence to be amplified and a 3′ “downstreamprimer” that hybridizes with the 3′ end of the sequence to be amplified.

A primer that is “perfectly complementary” has a sequence fullycomplementary across the entire length of the primer and has nomismatches. The primer is typically perfectly complementary to a portion(subsequence) of a target sequence. A “mismatch” refers to a site atwhich the nucleotide in the primer and the nucleotide in the targetnucleic acid with which it is aligned are not complementary. The term“substantially complementary” when used in reference to a primer meansthat a primer is not perfectly complementary to its target sequence;instead, the primer is only sufficiently complementary to hybridizeselectively to its respective strand at the desired primer-binding site.

The term “complementary” means that one nucleic acid is identical to, orhybridizes selectively to, another nucleic acid molecule. Selectivity ofhybridization exists when hybridization occurs that is more selectivethan total lack of specificity. Typically, selective hybridization willoccur when there is at least about 55% identity over a stretch of atleast 14-25 nucleotides, preferably at least 65%, more preferably atleast 75%, and most preferably at least 90%. Preferably, one nucleicacid hybridizes specifically to the other nucleic acid. See M. Kanehisa,Nucleic Acids Res. 12:203 (1984).

The term “stringent conditions” refers to conditions under which a probeor primer will hybridize to its target subsequence, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. In otherinstances, stringent conditions are chosen to be about 20° C. or 25° C.below the melting temperature of the sequence and a probe with exact ornearly exact complementarity to the target. As used herein, the meltingtemperature is the temperature at which a population of double-strandednucleic acid molecules becomes half-dissociated into single strands.Methods for calculating the T_(m) of nucleic acids are well known in theart (see, e.g., Berger and Kimmel (1987) Methods in Enzymology, vol.152: Guide to Molecular Cloning Techniques, San Diego: Academic Press,Inc. and Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual,2nd ed., vols. 1-3, Cold Spring Harbor Laboratory), both incorporatedherein by reference. As indicated by standard references, a simpleestimate of the T_(m) value can be calculated by the equation:T_(m)=81.5+0.41 (% G+C), when a nucleic acid is in aqueous solution at 1M NaCl (see e.g., Anderson and Young, “Quantitative FilterHybridization,” in Nucleic Acid Hybridization (1985)). Other referencesinclude more sophisticated computations which take structural as well assequence characteristics into account for the calculation of T_(m). Themelting temperature of a hybrid (and thus the conditions for stringenthybridization) is affected by various factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,and the like), and the concentration of salts and other components(e.g., the presence or absence of formamide, dextran sulfate,polyethylene glycol). The effects of these factors are well known andare discussed in standard references in the art, see e.g., Sambrook,supra, and Ausubel, supra. Typically, stringent conditions will be thosein which the salt concentration is less than about 1.0 M Na ion,typically about 0.01 to 1.0 M Na ion concentration (or other salts) atpH 7.0 to 8.3 and the temperature is at least about 30° C. for shortprobes or primers (e.g., 10 to 50 nucleotides) and at least about 60° C.for long probes or primers (e.g., greater than 50 nucleotides).Stringent conditions can also be achieved with the addition ofdestabilizing agents such as formamide.

The term “recombinant” when used with reference to a cell indicates thatthe cell replicates a heterologous nucleic acid, or expresses a peptideor protein encoded by a heterologous nucleic acid. Recombinant cells cancontain genes that are not found within the native (non-recombinant)form of the cell. Recombinant cells can also contain genes found in thenative form of the cell wherein the genes are modified and re-introducedinto the cell by artificial means. The term also encompasses cells thatcontain a nucleic acid endogenous to the cell that has been modifiedwithout removing the nucleic acid from the cell; such modificationsinclude those obtained by gene replacement, site-specific mutation, andrelated techniques.

A “heterologous sequence” or a “heterologous nucleic acid,” as usedherein, is one that originates from a source foreign to the particularhost cell, or, if from the same source, is modified from its originalform. Thus, a heterologous gene in a prokaryotic host cell includes agene that, although being endogenous to the particular host cell, hasbeen modified. Modification of the heterologous sequence can occur,e.g., by treating the DNA with a restriction enzyme to generate a DNAfragment that is capable of being operably linked to the promoter.Techniques such as site-directed mutagenesis are also useful formodifying a heterologous nucleic acid.

An “exogenous” species is refers to a species that is not normallypresent in a cell, but can be introduced into a cell by one or moregenetic, biochemical or other methods. Normal presence in the cell isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development is an exogenous moleculewith respect to a corresponding adult cell. An exogenous species can be,among other things, a small molecule, such as is generated by acombinatorial chemistry process, or a macromolecule such as a protein,nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein,polysaccharide, or any modified derivative of the above molecules, orany complex comprising one or more of the above molecules.

By contrast, an “endogenous” species is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. The term “operably linked” refers tofunctional linkage between a nucleic acid expression control sequence(such as a promoter, signal sequence, or array of transcription factorbinding sites) and a second polynucleotide, wherein the expressioncontrol sequence affects transcription and/or translation of the secondpolynucleotide.

The term “naturally occurring” as applied to an object means that theobject can be found in nature.

A “small molecule” means a synthetic molecule having a molecular weightof less than 1000 daltons, more typically 500 daltons or less. Suchmolecules include, for example, monosaccharides, polysaccharides,polypeptides, sterols, amino acids, lipids and nucleic acids.

The phrase “specifically binds” generally refers to binding of a ligandand an antiligand, or vice versa, with greater affinity and specificitythan to other components in the sample. Thus, the term refers to abinding reaction which is determinative of the presence of the ligand inthe presence of a heterogeneous population of other biologicalcompounds. Thus, under designated conditions, a specified ligand bindspreferentially to a particular antiligand and does not bind in asignificant amount to other molecules present in the sample. Typically,a molecule or ligand (e.g., an antibody) that specifically binds to anantiligand has an association constant of at least 10³ M⁻¹ or 10⁴ M⁻¹,sometimes 10⁵ M⁻¹ or 10⁶ M⁻¹, in other instances 10⁶ M⁻¹ or 10⁷ M⁻¹,preferably 10⁸ M⁻¹ to 10⁹ M⁻¹, and more preferably, about 10¹⁰ M⁻¹ to10¹¹ M⁻¹ or higher.

A difference is typically considered to be “statistically significant”if the difference is greater than the level of experimental error. Morespecifically, a difference is statistically significant if theprobability of the observed difference occurring by chance (the p-value)is less than some predetermined level. As used herein a “statisticallysignificant difference” refers to a p-value that is <0.05, preferably<0.01 and most preferably <0.001.

The term “label” refers to a molecule or an aspect of a molecule thatcan be detected by physical, chemical, electromagnetic and other relatedanalytical techniques. Examples of detectable labels that can beutilized include, but are not limited to, radioisotopes, fluorophores,chromophores, mass labels, electron dense particles, magnetic particles,spin labels, molecules that emit chemiluminescence, electrochemicallyactive molecules, enzymes, cofactors, and enzyme substrates. The term“detectably labeled” means that an agent has been conjugated with alabel or that an agent has some inherent characteristic (e.g., size,shape or color) that allows it to be detected without having to beconjugated to a separate label.

BRIEF DESCRIPTION OF THE DRAWINGS

In certain drawings, pumps are denoted with a group of three dashedlines and valves denoted by a single dashed line.

FIGS. 1A and 1B are illustrations of an elastomeric block and thearrangement of a control and flow channel therein.

FIG. 2A is a sectional view of an elastomeric block showing thedisposition of a flow and control channels with respect to one anotherin a valve and optional electrodes for actuating the valve.

FIG. 2B is a sectional view of an elastomeric block showing blockage ofa flow channel when a normally open valve is actuated.

FIGS. 3A and 3B are plan views illustrating the operation of anexemplary side-actuated valve structure.

FIGS. 4A and 4B show one example of a normally-closed valve structure.

FIG. 5 illustrates one arrangement of control and flow channels thatallow for selective blockage of certain flow channels.

FIGS. 6A and 6B illustrate one example of a peristaltic pump. FIG. 6A isa top schematic of the peristaltic pump. FIG. 6B is a sectionalelevation view along line 24B-24B in FIG. 6A.

FIGS. 7A-7C are top schematic views of different configurations of flowchannels that allow for mixing of solutions.

FIG. 8 is a top schematic view of an exemplary rotary pump.

FIG. 9 depicts an exemplary optical system for conducting singlemolecule or single cell measurements.

FIG. 10 is a cross-sectional view that illustrates the formation of aholding space within a flow channel upon actuation of valves in the flowchannel.

FIGS. 11A-11C depict different specific examples of cage structures.

FIG. 12 depicts an exemplary microfluidic device useful for conductinghigh throughput screening assays among other types of assays.

FIG. 13 depicts another exemplary microfluidic device useful forconducting high throughput screening assays among other types of assays.

FIG. 14 illustrates a multilayer soft lithography method forincorporating membranes into elastomeric fluidic devices.

FIG. 15 illustrates a sacrificial-layer encapsulation method forincorporating membranes into elastomeric fluidic devices.

FIGS. 16A and 16B depicts an exemplary microfluidic device incorporatinga membrane between flow channels.

FIGS. 17A-17C show exemplary microfluidic devices incorporatingmembranes that can be utilized in dialysis, filtration and capture andelution applications.

FIG. 18A illustrates elements of a separation module that can beincorporated into microfluidic devices or used as a stand alonemicrofluidic device.

FIG. 18B depicts an arrangement to prevent separation of elastomerlayers in a microfluidic device when flow channels are subjected to highpressures.

FIG. 19 depicts an exemplary microfluidic device useful for conductingcellular assays among other types of assays.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

Described herein are microfluidic devices and methods for conducting avariety of different assays, such as high throughput screening assays,cellular assays or assays involving cellular components, and syntheses,such as combinatorial syntheses. The microfluidic devices arecharacterized in part by including various components such as flowchannels, control channels, valves and/or pumps, at least some of whichare manufactured from elastomeric materials. This is in sharp contrastto conventional microfluidic devices that typically are based on siliconsubstrates (i.e., silicon chips).

The microfluidic devices in general include a main flow channel throughwhich a solution (e.g., a solution containing one or more analytes orcells, or solutions containing various assay agents) can flow, one ormore branch flow channels in fluid communication with the main flowchannel and one or more valves operatively disposed with respect to themain and/or branch flow channels. The valves comprise a control channelseparated from a flow channel by an elastomeric membrane or segment thatcan be deflected into or withdrawn from the flow channel upon actuationof the control channel (e.g., by applying pressure or a vacuum to thecontrol channel). When the membrane extends into the flow channel, itblocks solution flow through the channel. The microfluidic devices alsoinclude a detection section within the flow channel(s) and can furtherinclude one or more detectors positioned to detect a signal associatedwith a particular agent or event within the detection section of theflow channel.

In some of the microfluidic systems, each of the branch flow channelsincludes a pair of valves for retaining a volume of solution therebetween. Accordingly, the valves are positioned relative to one anothersuch that when the membranes of the two control channels associated withthe valve pair extend into the branch channel, the membranes form aholding space within the flow channel that is bounded by the extendedmembranes. This holding space generally is of a size appropriate to holdone or more particles, e.g., cells. With certain devices the membraneincludes teeth or a grate such that even when the valve is actuatedsolution containing assay reagents can flow into the space or a rinsesolution can flow through the space to carry away agents within thespace while the particle (s), e.g., cell (s), within the space areretained. The microfluidic devices also usually include one or moredetectors positioned to detect a signal associated with a particularagent or event within a detection section of the flow channel, which insome instances involves detection of a cell or other agent retained inthe holding space.

The branch flow channels can have a variety of different configurationsdepending upon the nature of the application. The flow channels areusually formed within an elastomeric material; in some instances, theentire microfluidic device is part of a monolithic elastomeric block. Inone embodiment of the present invention, the microfluidic devicesinclude a plurality of branch flow channels branching off of the mainflow channel. Each of the branch channels can include a set of valves asdescribed herein to provide a holding space within each branch flowchannel. In this way, different analyses or syntheses can be conductedin the different branch channels.

The microfluidic devices can include optional chambers or reservoirs.Such chambers or reservoirs can be positioned at the intersectionbetween the main flow channel and the branch flow channels to provide astorage site for solutions introduced through the main flow channeland/or the branch flow channels. Additionally, the microfluidic devicescan also optionally include one or more pumps for transporting solutionsalong and between the different flow channels. Certain pumps arecharacterized by including a plurality, preferably at least three,control channels that are separated from one of the flow channels by anelastomeric membrane that can be deflected into the flow channel whenactuated. By actuating the control channels in a staggered fashion, aperistaltic effect can be induced.

In some devices, the branch channels can include a pair of valves forretaining a volume of solution therebetween. Accordingly, the valves arepositioned relative to one another such that when the membranes of thetwo control channels of the valve pair extend into the branch flowchannel, the membranes form a holding space within the flow channel. Thebranch channels can also include optional mixers to mix and incubatesolutions. Certain mixers include a looped flow path that includes aplurality of peristaltic pumps of the type just described to flowsolution around the loop.

The microfluidic devices provided herein can be utilized in a number ofdifferent applications, for example, high throughput cellular assays. Bycontrollably introducing different solutions into the different branchchannels with the valves and transporting solutions between the mainflow channel and branch channels (e.g., using the pumps describedabove), a number of different analyses or syntheses can be performed atthe same time. Thus, the microfluidic devices can be used to conduct anumber of different types of assays, including, for example, bindingassays, cell reporter assays, toxicological assays, immunologicalassays, and single nucleotide polymorphism analysis.

The devices disclosed herein can be utilized to screen individualcompounds and libraries of compounds to identify those having a desiredeffect in various in vitro model systems. For example, assays utilizingthe microfluidic devices provided herein can be utilized to screenlibraries of compounds for those capable of fully or partiallyinhibiting reactions or processes that have undesirable consequences.For instance, libraries can be screened to identify compounds thatinhibit reactions or processes involved in the onset of disease orparticular symptoms associated with the disease (e.g., bacterial andviral infections, hereditary diseases and cancer). Alternatively,individual compounds and libraries of compounds can be screened toidentify particular compounds that activate or promoter reactions orprocesses of interest. Compounds showing activity in initial screeningcan then be subjected to other screens or modified and rescreened toidentify compounds suitable for formulation as pharmaceutical agents intreating the disease or symptoms associated with the disease underinvestigation.

In general such screening methods involve introducing certain assaycomponents (e.g., cells) into the different branch channels and thendifferent test agents (e.g., from a combinatorial library) areselectively introduced into the different branch flow channels such thatdifferent agents are delivered to different branch flow channels. Theability of the test agent to generate a particular response can bedetected by monitoring the holding area within the branch flow channels.Similarly, the valves and pumps of the microfluidic devices can beutilized to controllably react different reactants in the differentbranch flow channels to perform combinatorial syntheses.

II. Microfluidic Elements

A number of elements that are commonly utilized in the microfluidicdevices disclosed herein are described below. It should be recognizedthat these elements can be considered modules that can be combined indifferent ways to yield an essentially unlimited number ofconfigurations. Further, using the following elements or modules one cantailor the microfluidic device to include those elements useful for theparticular application (s) to be conducted with the device.

A. General

The microfluidic devices disclosed herein are typically constructed bysingle and multilayer soft lithography (MLSL) techniques and/orsacrificial-layer encapsulation methods. Both of these methods aredescribed in detail by Unger et al. (2000) Science 288: 113-116, in U.S.patent application Ser. No. 09/605,520, filed Jun. 27, 2000, in U.S.patent application Ser. No. 09/724,784, filed Nov. 28, 2000, and in PCTpublication WO 01/01025, all of which are incorporated herein in theirentirety for all purposes. The microfluidic devices provided herein caninclude a variety of different components that are described in detailinfra. These components can be arranged in a large number of differentconfigurations depending upon the particular application. The followingsections describe the general components that are utilized in thedevices; these sections are followed with exemplary configurations thatcan be utilized in various types of assays, such as cellular assays andhigh throughput screening.

Although the devices can be manufactured exclusively from elastomericmaterials, this is not a requirement. Thus, the devices need not bemonolithic in nature; hybrid devices fusing elastomers and othermaterials such as silicon, glass or plastic substrates can be utilized.As described in further detail below, the elastomeric materials can betailored to the particular application by modifying the internalsurfaces of the channels of the microfluidic device.

B. Channels

The channels through which solution is transported in the microfluidicdevices are typically formed at least in part, if not entirely, fromelastomeric compounds. Separated from the flow channels by anelastomeric membrane are control channels which can be actuated tocontrol or regulate solution flow through the flow channels. Asdescribed in greater detail below in the section on valves, actuation ofthe control channel (e.g., pressurization or pressure reduction withinthe flow channel) causes the elastomeric membrane separating the flowand control channel to be extended into the flow channel, thus forming avalve that blocks solution flow in the flow channel. Typically, the flowand control channels cross one another at an angle.

The flow and control channels can be manufactured from two primarytechniques. One approach is to cast a series of elastomeric layers on amicro-machined mold and then fuse the layers together. The secondprimary method is to form patterns of photoresist on an elastomericlayer in a desired configuration; in particular, photoresist isdeposited wherever a channel is desired. These two different methods offorming the desired configuration of flow and control channels, as wellas other details regarding channel dimensions and shape, are describedin considerable detail in PCT publication WO 01/01025, U.S. applicationSer. No. 09/605,520, filed Jun. 27, 2000, U.S. application Ser. No.09/724,784, filed Nov. 28, 2000, and by Unger et al. (2000) Science288:113-116, each of which is incorporated herein by reference in itsentirety.

C. Sample Inputs

There are a number of different options for introducing a solution intoa flow channel. One option is to simply inject solution into a flowchannel using a needle, for example. One can also pressurize a containerof solution to force solution from the container into a flow channel. Arelated approach involves reducing pressure at one end of a flow channelto pull solution into a distal opening in the flow channel.

Individual input/inlet lines can be formed that can be loaded manuallyusing single channel micropipettors. The microfluidic devices can besized according to industry size-specifications (e.g., footprint is127.76 0.12×85.47 0.12 mm) for plate readers and robotics and aredesigned to interface with generic multichannel roboticpipettors/samplers with standardized interwell spacings (pitch).Dimensional standards for these types of plate/devices are described athttp://www.tomtec.com/Pages/platstan.hmtl and http://www.sbsonline.com.Custom micropipettors that do not conform to this standard can also beutilized. In some systems, an electropipettor that is in fluidcommunication with a sample input channel is utilized. Micropipettors ofthis type are described, for example, in U.S. Pat. No. 6,150,180.

Inlets to the microfluidic devices disclosed herein can be holes orapertures that are punched, drilled or molded into the elastomericmatrix. Such apertures are sometimes referred to as “vias.” The vias canalso be formed using photoresist techniques. For example, metal etchblocking layers used in combination with patterning of photoresist masksand the use of solvents to remove etch blocking layers can be utilizedto create vias. Vertical vias between channels in successive elastomerlayers can be formed utilizing negative mask techniques. Vias can alsobe formed by ablation of elastomer material through application of anapplied laser beam. All of these techniques are described in greaterdetail in U.S. application Ser. No. 09/605,520.

Inlets can optionally be lined with couplings (e.g., made of Teflon) toprovide a seal with the pipette tips or syringe tip used to inject asolution.

As described further below, pumps formed from elastomeric materials canbe used to transport solution through the flow channels. For channels ofknown dimensions, one can precisely regulate the volume introducedthrough an inlet from based upon the number of strokes of the pump.

Any sample or solution that is chemically compatible with theelastomeric material from which the microfluidic device is fabricatedand which does not contain agents that are too large to pass through theflow channels can be introduced into the device. Examples of suitablesamples include, but are not limited to, aqueous buffers or mediacontaining cells, bacteria, viruses, phage, proteins, nucleic acids,small molecules, serum, whole blood or subtractions of blood, organicsolvents containing dissolved solutes, oils and mixtures of organic andaqueous solvents.

D. Valves

1. Structure

The valves of the microfluidic devices provided herein are formed ofelastomeric material and include a membrane or separating portion thatseparates a control channel and a flow channel. The valves have twogeneral designs: those that are typically open and those that arenormally closed. Valves that are typically open are actuated to blockflow through a flow channel by applying pressure to the control channel,thereby deflecting the membrane into the flow channel to restrict flow.In the case of valves that are normally closed, the membrane orseparating portion normally extends into the flow channel. However, uponreduction of pressure in the control channel relative to the flowchannel, the membrane/separating portion is pulled into the controlchannel, thus removing the blockage in the flow channel.

FIGS. 1A and 1B illustrate the general elements of a valve that istypically open. As can be seen, elastomeric structure 24 contains acontrol channel 32 overlying recess 21 formed from a raised portion of amold. When the recess in this elastomeric structure is sealed at itsbottom surface to planar substrate 14, recess 21 forms a flow channel30. As can be seen in FIG. 1B and FIG. 2A, flow channel 30 and controlchannel 32 are preferably disposed at an angle to one another with asmall membrane 25 of elastomeric block 24 separating the top of flowchannel 30 from the bottom of control channel 32. While these figuresshow control channels that extend across the device, it should beunderstood that this need not be the case. The control channel can be arecess sufficiently large such that the membrane is able to provide thedesired level of blockage in the flow channel. FIG. 2B illustrates thesituation for a normally open elastomeric valve structure 200 in whichthe valve has been actuated and the flow channel is blocked. Inparticular, the structure includes a control channel 120 formed withinone elastomeric layer 110 that overlays another elastomeric layer 128which includes a flow channel 126. Elastomeric layer 110 is attached tosubstrate 130. Because the control channel has been pressurized, themembrane 122 separating the control channel 120 and the flow channel 126is deflected down into the flow channel 126, thereby effectivelyblocking solution flow therethrough. Once pressure is released, membrane122 deflects back up from the flow channel 126 to allow solution flow.

In certain devices, planar substrate 14 is glass. The transparentproperties of glass can be useful in that it allows for opticalinterrogation of elastomer channels and reservoirs. Alternatively, theelastomeric structure can be bonded onto a flat elastomer layer, therebyforming a permanent and high-strength bond. This can prove advantageouswhen higher back pressures are generated. Hence, the choice of substrateupon which a flow channel is formed (e.g., glass or elastomer) dependsin part on the type of detection utilized, as well as the structuralrequirements of the device.

While the valve shown in FIGS. 1B and 2 involve a system in which acontrol channel overlays a flow channel, different configurations can beutilized. For example, FIGS. 3A and 3B illustrate a side-actuated valve.More specifically, FIG. 3A shows side-actuated valve structure 4800 inan unactuated position. Flow channel 4802 is formed in elastomeric layer4804. Control channel 4806 abutting flow channel 4802 is also formed inelastomeric layer 4804. Control channel 4806 is separated from flowchannel 4802 by elastomeric membrane portion 4808. A second elastomericlayer (not shown) is bonded over bottom elastomeric layer 4804 toenclose flow channel 4802 and control channel 4806. FIG. 3B showsside-actuated valve structure 4800 in an actuated position. In responseto a build up of pressure within control channel 4806, membrane 4808deforms into flow channel 4802, blocking flow channel 4802. Upon releaseof pressure within control channel 4806, membrane 4808 relaxes back intocontrol channel 4806 and opens flow channel 4802.

As noted above, the valves can also have a normally closedconfiguration. FIG. 4A illustrates one example of a normally-closedvalve 4200 in an unactuated state. Flow channel 4202 and control channel4204 are formed in elastomeric block 4206. Flow channel 4202 includes afirst portion 4202 a and a second portion 4202 b separated by separatingportion 4208. Control channel 4204 overlies separating portion 4208. Asshown in FIG. 4A, in its relaxed, unactuated position, separatingportion 4208 remains positioned between flow channel portions 4202 a and4202 b, interrupting flow channel 4202. FIG. 4B shows a cross-sectionalview of valve 4200 wherein separating portion 4208 is in an actuatedposition. When the pressure within control channel 4204 is reduced tobelow the pressure in the flow channel (for example by vacuum pump),separating portion 4208 experiences an actuating force drawing it intocontrol channel 4204. As a result of this actuation force, membrane 4208projects into control channel 4204, thereby removing the obstacle tosolution flow through flow channel 4202 and creating a passageway 4203.Upon elevation of pressure within control channel 4204, separatingportion 4208 assumes its natural position, relaxing back into andobstructing flow channel 4202.

It is not necessary that the elastomeric layers that contain the flowand control channels be made of the same type of elastomeric material.For example, the membrane that separates the control and flow channelscan be manufactured from an elastomeric material that differs from thatin the remainder of the structure. A design of this type can be usefulbecause the thickness and elastic properties of the membrane play a keyrole in operation of the valve.

2. Options for Actuating Valves

A variety of approaches can be utilized to open or close a valve. If avalve is actuated by increasing pressure in a control channel, ingeneral this can be accomplished by pressurizing the control channelwith either a gas (e.g., air) or a fluid (e.g., water or hydraulicoils). However, optional electrostatic and magnetic actuation systemscan also be utilized. Electrostatic actuation can be accomplished byforming oppositely charged electrodes (which tend to attract one anotherwhen a voltage differential is applied to them) directly into themonolithic elastomeric structure. For example, referring once again toFIG. 2, an optional first electrode 70 (shown in phantom) can bepositioned on (or in) membrane 25 and an optional second electrode 72(also shown in phantom) can be positioned on (or in) planar substrate14. When electrodes 70 and 72 are charged with opposite polarities, anattractive force between the two electrodes will cause membrane 25 todeflect downwardly, thereby closing the “valve” (i.e., closing flowchannel 30).

Alternatively, magnetic actuation of the flow channels can be achievedby fabricating the membrane separating the flow channels with amagnetically polarizable material such as iron, or a permanentlymagnetized material such as polarized NdFeB. Where the membrane isfabricated with a magnetically polarizable material, the membrane can beactuated by attraction in response to an applied magnetic field.

Optional electrolytic and electrokinetic actuation systems can also beutilized. For example, actuation pressure on the membrane can begenerated from an electrolytic reaction in a recess overlying themembrane. In such an embodiment, electrodes present in the recess areused to apply a voltage across an electrolyte in the recess. Thispotential difference causes an electrochemical reaction at theelectrodes and results in the generation of gas species, thereby givingrise to a pressure differential in the recess. Alternatively, actuationpressure on the membrane can arise from an electrokinetic fluid flow inthe control channel. In such an embodiment, electrodes present atopposite ends of the control channel are used to apply a potentialdifference across an electrolyte present in the control channel.Migration of charged species in the electrolyte to the respectiveelectrodes can give rise to a pressure differential. Finally, valves canbe actuated the device by causing a fluid flow in the control channelbased upon the application of thermal energy, either by thermalexpansion or by production of gas from liquid. Similarly, chemicalreactions generating gaseous products may produce an increase inpressure sufficient for membrane actuation.

3. Options for Selectively Actuating Valves

In order to facilitate fabrication and to reduce the number of controlchannels in a microfluidic device, often a control channel overlays anumber of flow channels. In such instances, pressurization of such acontrol channel could cause blockage of all the flow channels. Often itis desired to block only selected flow channels, rather than all theflow channels which a control channel abuts. Selective actuation can beachieved in a number of different ways.

One option illustrated in FIG. 5 is to control the width of the controlchannels 5004, 5006 at the point at which they extend across the flowchannels 5002A and 5002B. In locations where the control channels arewide 5004A, 5006A, pressurization of the control channel 5004, 5006causes the membrane separating the flow channel and the control channelto depress significantly into the flow channel 5002A, 5002B, therebyblocking the flow passage therethrough. Conversely, in the locationswhere the control line is narrow 5004B, 5006B, the membrane separatingthe channels is also narrow. Accordingly, the same degree ofpressurization will not result in membrane becoming depressed into theflow channel 5002A, 5002B. Therefore, fluid passage thereunder will notbe blocked.

The same general effect can be obtained by varying the width of the flowchannel relative to the control channel. Incorporation of an elastomericsupport in the section of the flow channel opposite the membrane that isdeflected into the flow channel can also prevent complete stoppage ofsolution flow.

Valves in certain of the figures are represented by single dashed linesif the valve can be utilized to block solution flow through the flowchannel. A control channel that crosses a flow channel but which doesnot act to block the flow channel (for the reasons just described) isrepresented by a solid arch that arches over a flow channel.

Various other methods of actuating valves are described in the aboveincorporated U.S. and PCT applications.

E. Pumps

The pumps integrated within the microfluidic devices described hereincan be formed from a plurality of control channels that overlay a flowchannel. A specific example of a system for peristaltic pumping is shownin FIGS. 6A and 6B. As can be seen, a flow channel 30 has a plurality ofgenerally parallel control channels 32A, 32B and 32C passing thereover.By pressurizing control line 32A, flow F through flow channel 30 is shutoff under membrane 25A at the intersection of control line 32A and flowchannel 30. Similarly, (but not shown), by pressurizing control line32B, flow F through flow channel 30 is shut off under membrane 25B atthe intersection of control line 32B and flow channel 30, etc. Each ofcontrol lines 32A, 32B, and 32C is separately addressable. Therefore,peristalsis can be actuated by the pattern of actuating 32A and 32Ctogether, followed by 32A, followed by 32A and 32B together, followed by32B, followed by 32B and C together, etc. Pumps of this type are denotedin shorthand form in certain of the figures with a series of threeparallel dashed lines.

External pumps can also be connected to a flow channel to transportsolutions through a channel. Alternatively, a vacuum can be applied to aflow channel to direct fluid flow toward the region of reduced pressure.

F. Mixer Units

Units for mixing one or more fluids introduced the flow channels can behave a variety of different configurations. One simple way to rapidlymix fluids is simply to have two solutions flow into one another in a Y-or T-shaped junction. This is illustrated in FIG. 7A where solution Aflows through section 702A of flow channel 702, with solution B flowingthrough section 702B. Optional control channels can overlay each sectionto control solution flow into the junction if desired. At theintersection of flow channel 702 with flow channel 704, solutions A andB are mixed to form solution C. Similarly, in the Y-shaped junctiondepicted in FIG. 7B, solution A flowing through branch channel 708 andsolution B flowing through channel 710 converge at flow channel 712 toform solution C.

Another mixing option is to flow solutions repeatedly back and forthbetween two reservoirs or chambers. Such an arrangement is depicted inFIG. 7C where a flow channel 720 include two expanded regions 722A, 722Bto form two reservoirs in which solution can collect. Control channels724A, 724B, and 724C can be placed on either side of the reservoirs722A, 722B to control fluid flow therebetween. Solution A can beintroduced into reservoir 722A and solution B introduced into reservoir722B. By opening valve 724B located between the two reservoirs 722A,722B, the two solutions can be mixed. Mixing and incubation can continueas the solutions are moved back and forth between the two reservoirs722A, 722B. One way this can be accomplished is to open valve 724A andthen move solution from reservoir 722A into reservoir 722B. Thedirection of flow is then reversed by closing valve 724A, opening valve724C, and then transporting solution from reservoir 722B back toreservoir 722B.

A rotary pump can also be utilized to mix and incubate solutions. Thissystem involves the use of one or more pumps to flow solution around acircular flow channel. FIG. 8 depicts one such pump 800. This particularrotary pump 800 includes a closed loop flow channel 802. Twoinlet/outlet flow channels 804A, 804B are in fluid communication withthe circular flow channel 802. Valves 806A, 806B are located within theinlet/outlet flow channels 804A, 804B to control fluid flow into and outof the circular flow channel 802. Pumps 808A, 808B of the designdescribed supra (i.e., a plurality of control channels that overlay theflow channel) are located across from one another in the closed loopflow channel 802. The rotary pump 800 can be positioned adjacent atemperature control device 810 to regulate temperature of the mixture asit is pumped through the rotary pump.

In operation, valves 806A, 806B are both initially closed. Valve 806A isthen opened to allow solution A to enter the circular flow channel 802.Valve 806A is then closed and valve 806B opened to allow solution B toenter the circular flow channel 802. Valve 804B is then also closed andsolutions A and B mixed and incubated by circulating the mixture underthe action of pumps 808A, 808B. After the solutions have been mixed andincubated for the desired length of time, either of (or both) valves806A, 806B) are opened and the mixture withdrawn from the circular flowchannel 802.

Although the particular pump illustrated in FIG. 8 utilizes a circularflow channel, the actual shape of the flow channel can be essentiallyany shape so long as the loop is a closed loop. Thus, the flow channelcan be circular, oblong, or serpentine in shape. Furthermore, the orderand timing of solution introduction into the loop can also be varied.

G. Detection Units

1. General

The microfluidic devices provided can be utilized in combination with awide variety of detection methodologies. Detection can involve thedetection of particular events and/or particular entities such asparticles, beads and cells. Detection can be accomplished usingdetection methodologies in use in flow cytometry and liquidchromatography. Examples of particular detection methods useful with thepresent microfluidic devices include, but are not limited to, lightscattering, multichannel fluorescence detection, UV and visiblewavelength absorption, luminescence, differential reflectivity, andconfocal laser scanning. Applications can also utilize scintillationproximity assay techniques, radiochemical detection, fluorescencepolarization, fluorescence correlation spectroscopy (FCS), time-resolvedenergy transfer (TRET), fluorescence resonance energy transfer (FRET)and variations such as bioluminescence resonance energy transfer (BRET).Additional detection options include electrical resistance, resistivity,impedance, and voltage sensing.

Alternatively, fluids can be transported directly to a mass spectrometerinterface (e.g., electrospray ionization, ESI) or directed onto a matrixof flow interface for analysis by Matrix-Assisted Laser DesorptionIonization-Time of Flight Mass Spectrometry (MALDI-TOF). Detection andmeasurement based on these methodologies can be used to sort cells ordirect cells, beads or particles into new locations on or off themicrofluidic device.

The term “detection section,” “detection region,” and other like termsrefer to the portion of the microfluidic device at which detectionoccurs. In general, the detection section can be at essentially anypoint along one of the flow channels or at an intersection of flowchannels. The detection region is in communication with one or moremicroscopes, diodes, light stimulating devices (e.g., lasers),photomultiplier tubes, processors and combinations of the foregoing,which cooperate to detect a signal associated with a particular event oragent. In some instances, the detection section is coincident with aholding space; in other instances the detection section includes arotary pump, while in still other instances, the detection section islocated adjacent to a rotary pump or holding space, with detectionoccurring following complete mixing of solutions in the mixer or releaseof solution from the holding space.

Often the signal being detected is an optical signal that is detected inthe detection section by an optical detector. The optical detector caninclude one or more photodiodes (e.g., avalanche photodiodes), afiber-optic light guide leading, for example, to a photomultiplier tube,a microscope, and/or a video camera (e.g., a CCD camera).

The optical detector can be microfabricated within the microfluidicdevice, or can be a separate element. If the optical detector exists asa separate element and the microfluidic device includes a plurality ofdetection sections, detection can occur within a single detectionsection at any given moment. In other instances, an automated system isutilized which scans the light source relative to the microfluidicdevice, scans the emitted light over a detector, or includes amultichannel detector. For example, the microfluidic device can beattached to a translatable stage and scanned under a microscopeobjective. The acquired signal is routed to a processor for signal.interpretation and processing. Arrays of photomultiplier tubes can alsobe utilized. Additionally, optical systems that have the capability ofcollecting signals from all the different detection sectionssimultaneously while determining the signal from each section can beutilized.

In some instances, the detection section includes a light source forstimulating a reporter that generates a detectable signal. The type oflight source utilized depends in part on the nature of the reporterbeing activated. Suitable light sources include, but are not limited to,lasers, laser diodes and high intensity lamps. If a laser is utilized,the laser can be utilized to scan across a set of detection sections, orlaser diodes can be microfabricated into the microfluidic device itself.Alternatively, laser diodes can be fabricated into another device thatis placed adjacent to the microfluidic device being utilized to conductan assay such that the laser light from the diode is directed into thedetection section.

In some instances in which external radiation and/or detector areutilized, an substrate optically transparent at the wavelength beingmonitored is used to cover the detection section. However, byappropriate selection of elastomeric materials, monolithic elastomericdevices can still be utilized in conjunction with a wide variety ofexternal optical detection methods.

Detection can involve a number of non-optical approaches as well. Forexample, the detector can also include, for example, a temperaturesensor, a conductivity sensor, a potentiometric sensor (e.g., pHelectrode) and/or an amperometric sensor (e.g., to monitor oxidation andreduction reactions).

In certain methods, solutions are transported from the microfluidicdevice to a separate external device for further analysis. The externaldevice can be any of a number of analytical devices such as UV/VIS, IR,NMR and/or ESR spectrometers; chromatographic columns (e.g., HPLC);and/or mass spectrometry, for example.

2. Optical Microscope

Certain methods utilize an optical microscope to examine the differentdetection sections in the different flow channels. The objective lens ofthe microscope is directed towards the detection section. Typically, amercury arc lamp or argon laser is utilized as the light source. Themicrofluidic device can be mounted on a translation stage such that thevarious detection sections can be positioned by translation over theobjective lens. Additional details regarding the use of microscopes withmicrofluidic devices similar to those described herein are provided inPCT publication WO 99/61888.

3. Fluorescent Detection Systems

Detection methodologies that can be utilized in the screening processinclude, and are not limited to: (1) fluorescence intensity, (2)fluorescence polarization, (3) fluorescence resonance energy transfer(FRET), (4) heterogeneous time resolved fluorescence (HTRF) ortime-resolved energy transfer (TRET), (5) Fluorescence correlationspectroscopy (FCS) and related techniques (such as fluorescenceintensity distribution analysis (FIDA). (see, e.g., Pope et al. (1999)Drug Discovery Today 4: 350-362; Kask et al. (1999) Proc. Natl. Acad.Sci. USA. 96: 13756-61; Moore et al. (1999) J. Biomol. Screening 4:335-353; and Auer et al. (1999) Drug Discovery Today 3: 457-465). A moredetailed discussion of these detection options follows.

Fluorescence intensity: Measurement of the intensity of fluorescence ofa sample provides a direct measurement of fluorophore concentration.This technique is often used in enzyme assays, where an enzyme activityis measured using a non-fluorescent substrate that is converted to afluorescent product by the action of the enzyme (i.e., a fluorogenicsubstrate). Other assays that measure fluorescence directly includecalcium binding assays, in which the fluorescence of the calcium bindingdye is significantly increased upon binding calcium. Thus, the detectorin certain systems is an instrument able to detect fluorescenceintensity from the detection section of the microfluidic device.

Fluorescence polarization: Fluorescence polarization (FP) is anothercommon detection technique that can be utilized with the microfluidicdevices provided herein. The theory of FP is that when a fluorophore isexcited with polarized light, the emitted light is also polarized. Thisoccurs because excitation is dependent upon the orientation of thefluorophore dipole to the excitation beam. The emitted light isdepolarized upon rotational diffusion of the fluorophore. For a smallmolecule fluorophore, this occurs rapidly and the emitted light isisotropic. Changes in the rotational diffusion time of a smallfluorophore occur when it becomes bound to a much larger molecule andlead to measurable anisotropy in the emitted light. Thus, FP can beutilized in a wide variety of assays in which in certain circumstances afluorescently labeled agent is part of a large molecule that tumblesrelatively slowly, whereas in other circumstances the labeled agent isfree in solution and able to tumble more rapidly. Examples of suchassays include assays involving binding of a labeled ligand to acell-surface receptor, ligand/antiligand binding (e.g., ligand/receptorbinding) and a labeled protein substrate and a labeled cleavage product.

Fluorescence polarization is determined by measuring the vertical andhorizontal components of fluorophore emission following excitation withplane polarized light. Light from a monochromatic source (at anappropriate excitation wavelength) passes through a vertical polarizingfilter to excite fluorescent molecules in the sample. Only thosemolecules that are orientated in the vertically polarized plane absorblight, become excited, and subsequently emit light. The emission lightintensity is measured both parallel and perpendicular to the excitinglight. The fraction of the original incident, vertical light intensitythat is emitted in the horizontal plane is a measure of the amount ofrotation the fluorescently labeled molecule has undergone during theexcited state, and therefore is a measure of its relative size. Thus,the detector used to monitor FP in the microfluidic device includes theelements necessary to make the foregoing measurements (see FIG. 9 anddiscussion infra). A number of commercially-available FP instruments canbe used in conjunction with the present microfluidic devices (e.g.,systems from Panvera Corp). Additional guidance regarding FP detectionis provided, for example, by Chen et al. (1999) Genome Research 9:492-8; and in U.S. Pat. No. 5,593,867 to Walker et al.

Fluorescence resonance energy transfer (FRET): This technique isdependent upon non-radiative transfer between two fluorophores (a donorand an acceptor) that occurs when they come into close proximity (<5nm). The efficiency of transfer highly dependent upon the distancebetween the fluorophores, their physical properties and the spectraloverlap between them. Under FRET conditions, excitation at the donorexcitation maximum is efficiently transferred to the acceptor andemitted at the acceptor emission wavelength. This property can beexploited in many different types of assays that can either bringfluorophores together (increased FRET) or separate them (decreasedFRET). Thus, FRET assays can be conducted by detecting an increase inthe fluorescence intensity of the acceptor and a decrease influorescence intensity of the acceptor. Alternatively, changes in theratio of emission at the donor emission maximum to emission at theacceptor maximum can be used to follow increases or decreases in FRET.The present microfluidic devices can be utilized in FRET assays inconjunction with commercially-available fluorescent readers. Thesesystems include a source to activate the acceptor fluorophore and thendetect alterations in the emissions from the donor and/or acceptorfluorophore.

A number of fluorophores suitable for conducting FRET assays are known.Specific examples include, 6-carboxy fluorescein (FAM),5&6-carboxyrhodamine-110 (R110), 6-carboxyrhodamine-6G (R6G),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine(ROX), 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE), ALEXAFluor™, Cy2, Texas Red and Rhodamine Red. Additional fluorescent dyesavailable from Applied Biosystems Division of Perkin Elmer Corporation(Foster City, Calif.) include,6-carboxy-2′,4,7,7′-tetrachlorofluorescein (TET),6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein (HEX),5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein (ZOE), NAN, NED;fluorophores available from Amersham Pharmacia Biotech (Piscataway,N.J.) include, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.5.

Further guidance regarding the selection of donor and acceptor pairsthat can effectively be used in FRET-based assays include: FluorescenceSpectroscopy (Pesce et al., Eds.) Marcel Dekker, New York, (1971); Whiteet al., Fluorescence Analysis: A Practical Approach, Marcel Dekker, NewYork, (1970); Berlman, Handbook of Fluorescence Spectra of AromaticMolecules, 2nd ed., Academic Press, New York, (1971); Griffiths, Colourand Constitution of Organic Molecules, Academic Press, New York, (1976);Indicators (Bishop, Ed.). Pergamon Press, Oxford, 19723; and Haugland,Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes,Eugene (1992).

Another option is to use various fluorescent proteins. Examples includegreen fluorescent protein (GFP), blue fluorescent protein (BFP), yellowfluorescent protein and ds Red (a red fluorescent protein).

Time-resolved techniques: A variety of time-resolved fluorescenttechniques can be utilized. One such technique is heterogeneoustime-resolved fluorescence (HTRF) or time resolved energy transfer(TRET). This method uses fluorescence resonance energy transfer betweentwo fluorophores. The most commonly used donor is europium cryptate(EuK), which absorbs light at 337 nm and emits at 620 nm. Other commonlyused long-lived donors are lanthanates (Ln³⁺). EuK can transmit thisenergy in a non-radiative fashion to an appropriate acceptor, such asXL665 (a modified allophycocyanin) when the acceptor-donor pair are inclose proximity (<5-10 nm). When excited at 620 nm, XL665 emits lightwith a slow decay at 665 nm. Detection is performed after a delay(usually ˜50 μs) as the measured ratio of fluorescence at 665 nm tofluorescence at 620 nm (F₆₆₅/F₆₂₀). The advantage of using anacceptor-donor pair with long lifetimes is that background fluorescencedecays more rapidly than the desired signal, and consequently HTRF isextremely sensitive.

Fluorescence correlation spectroscopy (FCS): This method is based uponthe recognition that as a fluorescently labeled molecule passes througha confocal laser beam and is excited, it emits photons of fluorescentlight. The length of each photon burst is dependent upon the time spentin the confocal beam, and is diffusion controlled. By measuring the timeassociated with each burst, diffusion coefficients can be calculated,allowing discrimination of fluorescent molecules, such as bound and freespecies in a solution. Quantitation of free and bound ligand thereforeallows determination of absolute concentrations of fluorophores anddegree of binding. FCS is insensitive to miniaturization and thereforeuseful for implementation in microfluidic devices. When utilized withthe present devices, a confocal laser is oriented such that the beam itemits is directed towards the detection section. The fluorescentdetector is positioned to receive the photons of emitted light receivedfrom the detection section.

Ligands in Detection Section: Certain detection methods involveimmobilizing an antiligand within the detection section. In this way,ligands that specifically bind to the antiligand can be captured anddetected within the detection section. Often the antiligand is animmunological agent such as an antibody. The use of this mode ofdetection is described in further detail infra.

4. Single Molecule and Single Cell Measurements

Certain detection units that can be utilized with the systems describedherein permit the detection and measurement of single molecules orcells. This capability can enable one to study processes that might notbe apparent when making measurements of ensemble averages of populationsof molecules or cells. In particular, such measurements allowobservation of subpopulations of events within apparently homogeneoussystems, and the analysis of dynamic events occurring on different timescales that would be lost upon averaging (see, e.g., Ishii, Y. andYanagida, T. (2000) Single Mol. 1: 5-16 and Weiss S. (1999) Science 283:676-1683. Fluorescence Correlation Spectroscopy (FCS; described supra)is one example of an intrinsically single molecule detection techniquein which such detection units are useful. However, with standard optics,one can readily detect events at the single molecule or single celllevel in essentially all of the modes described above (fluorescenceintensity, fluorescence polarization, fluorescence resonance energytransfer (FRET), and fluorescence correlation spectroscopy (FCS)).Optical systems for the detection of single DNA molecules and cells inmicrofluidic devices are described in PCT Publication WO 99/61888, whichis incorporated by reference in their entirety for all purposes.

The optical arrangement discussed in these publications can be modifiedto determine multiple signals from a detection section. For instance,using commercially available components (such as described athttp://www.newport.com; 2001 Catalog), polarization optics can beutilized to prepare a device in which relative polarized signal strengthis determined. One example of such a system is illustrated in FIG. 9. Asshown, the optical system includes a laser that generates a laser beamthat is directed towards the detection section of the microfluidicdevice by a dichroic mirror. Light emission from species (e.g.,fluorescently labeled compounds) within the detection section arecollected by a lens positioned adjacent to the detection section. Thecollected light passes through the dichroic mirror and is then split bya polarizing beam splitter cube into a plurality of light beams. Thesplit polarized light is focused with focusing optics and the resultingsignals are detected with a plurality of detectors, the system includingone detector for each polarization component separated by the polarizingbeamsplitter. With an arrangement such as this, one can measure signalintensity for different components of polarized light. Thus, in afluorescence polarization experiment, for example, one can utilize suchan arrangement to distinguish between an unbound molecule and the sametype of molecule bound to a large species such as a cell. Because anunbound small molecule can tumble rapidly in solution, it emitsnon-polarized light. However, if the molecule becomes bound to the largespecies, the rate of tumbling slows dramatically and results in theemission of polarized light. One can detect a species that inhibitsbinding by contacting the complex with the test species and thendetermining whether there is a decrease in the emission of polarizedlight. Various modifications of the optical system shown in FIG. 9 canbe utilized. For instance, a polarized rotator (½ wave plate; seeNewPort catalog) positioned in the excitation beam prior to the dichroicmirror, allows for variation of the polarization of the excitation lightfor purposes of measurement optimization. In another arrangement, asecond PMT detector and appropriate dichroic mirrors and filters arepositioned so as to detect the ratio of coupled (FRET) or uncoupledemissions at different wavelengths (see, e.g., Weiss S. (1999) Science283: 1676-1683).

III. High Throughput Screening Systems A. General

As indicated supra, the foregoing elements or modules can be combined ina large number of configurations and utilized in a wide variety ofapplications. Exemplary designs useful for conducting certain types ofassays are described in this section. It should be understood, however,that the microfluidic devices of the present invention are not limitedto these particular configurations.

In general, the high throughput screening devices typically include aplurality of branch flow channels that intersect with, and are in fluidcommunication with, a main flow channel. The devices are designed suchthat common assay components (i.e., components utilized in each assay inthe various branch flow channels such as cells, enzyme cofactors andbuffers) are introduced into the main flow channel while blocking flowthrough the various branch flow channels. Once the common assaycomponents have been introduced into the main flow channel, flow throughthe main flow channel is blocked. Different test agents or samples aresubsequently introduced into the different branch flow channels,typically such that different test agents are introduced into differentbranch flow channels. Signal detection occurs within a detection region.

The microfluidic device can optionally include a holding space (e.g., apen or cage). With such devices, one can initially introduce differentsamples into the branch flow channels and store a portion of the samplein the branch flow channels in the holding space. Assay componentscommon to each assay are then introduced into the main flow channel.Retained solution in the branch flow channels is subsequently reactedwith the assay components in the main flow channel. Typically, this isdone by transporting retained solution back to the main flow channel forreaction with assay components therein.

Regardless of the particular design, a large number of test agents canbe rapidly screened within a short time period because separatereactions can simultaneously be conducted in each of the flow channels.

Each branch flow channel can also optionally includes a mixer and/or apump. The mixer can be used to mix and circulate solutions, typicallyafter all the various solutions have been combined. The pumps areutilized for transporting fluids within the branch flow channel. Themixer and pump typically are elastomeric structures of the designs setforth supra.

B. Exemplary Devices

1. Configuration with a Single Main Flow Channel

A specific example of a microfluidic device that can be utilized in suchhigh throughput screens is illustrated in FIG. 12. The device 800includes a main flow channel 802 that is in fluid communication with aplurality of branch flow channels 842A, 842B, 842C. Each of the branchflow channels 842A, 842B, 842C intersect with the main flow channel 802at a chamber (also called a capacitor or well) 820A, 820B, 820C that issized to receive and store solution flowing within the main flow channel802 and the branch flow channels 842A, 842B, 842C. The chambers 820A,820B, 820C can include a depression formed into the elastomericsubstrate into which the channels are formed. Other chambers consist ofan expanded region of a flow channel. Each branch flow channel includesan optional mixer unit 826A, 826B, 826C, a detection region 828A, 828B,828C and a pump 830A, 830B, 830C. Typically, the detection section 828A,828B, 828C is located between the mixer unit 826A-C and pump 830A-C. Inthis way the pumps 830A-C can pull solution through the branch flowchannel into the mixer unit 826A-C where solutions can be mixed and/orincubated and then, at the appropriate time, transported to thedetection section 828A-C. The mixer unit 826A-C and pump, 830A-C can beof the structure of described supra. The detection region 828A-C can bea section of the branch flow channel 842A-C that a detector is orientedto monitor. If an optical detector is utilized, then this section caninclude a material that is substantially optically transparent at thewavelength being monitored. The detection section 828A-C can alsooptionally include a holding space such as a pen or cage to retain avolume of solution that may also include cells or various supports(e.g., beads or particles).

In operation, to introduce assay components common to all the differentassays, valve 806 is opened and assay solution introduced into the mainflow channel 802 via inlet 804. Valves 810, 814, 818 in the branch flowchannels typically are closed to block solution flow therethrough. Assaysolution accumulates in the chambers 820A, 820B, 820C located along themain flow channel 802. An optional pump (not shown) located downstreamof the last branch flow channel can be utilized to control the rate ofsolution flow through the main flow channel 802. Agents to be screenedare introduced by closing valve 806 in the main flow channel 802 torestrict solution flow through the main flow channel while opening thebranch flow channel valves 808, 812, 816.

Test agents or samples introduced into the branch flow channels 842A-Cinitially collect in the chambers 820A-C. Mixtures can beincubated/stored in the chambers 820A-C. Alternatively, valves810,814,818 in the branch flow channels 842A-C are opened and mixturesfrom the chambers 820A-C withdrawn into the respective branch flowchannels under the action of pumps 830A-C. Mixtures can be introducedinto the mixing units 826A-C to allow further mixing and/or incubation.The resulting mixture is then pumped in a downstream direction towardsthe detection regions 828A-C where the solution can be assayed by anappropriate detector (not shown). Assays can be discrete, single timepoint (endpoint) assays, or involve sampling the mixture from thechamber or mixing units at various time periods (kinetic assays). Insome assays, pumps and branch flow valves are operated such thatcomponents can be moved back and forth within a branch flow channel(e.g., between a holding space or mixer and the chambers), therebypromoting sequestration and mixing at the desired time.

As indicated supra, another option is to initially introduce thedifferent test agents into the branch flow channels 842A-C. A portion ofthe solution containing the test agent is retained in the branch flowchannels in a holding region located within the detection regions828A-C. Common assay agents can then be introduced into the main flowchannel 802 to introduce assay components into chambers 820A-C. Retainedtest agents in each of the branch flow channels 842A-C can then beflowed back to the chambers 820A-C to be mixed with the assaycomponents. The resulting mixtures in the chambers 820A-C are thentransported under action of the pumps 842A-C to their respectivedetection sections 828A-C for signal detection by a detector.

2. Configuration with Multiple Main Flow Channels

Another specific example of a system that can be used, for example, inhigh throughput screening applications is illustrated in FIG. 13. As canbe seen, this particular configuration is similar to the configurationjust described and illustrated in FIG. 12, but differs in that itincludes a plurality of main flow channels, indicated in FIG. 13 as A, Band C. Although this particular figure shows only three main flowchannels (A, B, and C), it should be understood that relatedconfigurations including additional flow channels can also be utilizedand are included in the present invention. Likewise, it should also berecognized that additional branch flow channels (indicated in FIG. 13 as1, 2, 3) can be included. The inlet to each of the main flow channels,can include multiple injection ports, indicated in FIG. 13 as port (a)and (b). Of course additional ports could be include at each inlet.

In other respects, however, the configuration is similar to that shownin FIG. 12. For example, the device typically includes chambers at eachintersection between a main flow channel (A, B, C) and branch channel(1, 2, 3). Additionally, each branch flow channel (1, 2, 3) can includean optional rotary mixer, detection region, and/or a pump. Detection canoccur at each chamber and/or downstream of the main flow channels at adetection region separate from the chambers.

One situation in which the particular configuration depicted in FIG. 13is useful is when a number of different assay components are to beadded. In such situations, the multiple main flow channels can beutilized to separately introduce different assay components. This can beparticularly useful when certain assay components need to be incubatedbefore mixing with another assay component. For example, in a screen toidentify compounds that inhibit the interaction between a protein and aligand, cells expressing the receptor of interest can be introduced inmain flow channel A such that cells are deposited in the differentchambers along flow channel A. Different potential inhibitors can thenbe introduced into the different branch flow channels (1, 2, 3) andallowed to contact the cells in the respective chamber in the branchflow channel. The potential inhibitors and cells are then allowed toincubate to allow for the potential inhibitor to bind to the receptor.Subsequently, a known ligand for the receptor can be introduced intomain flow channel B and into the chambers along flow channel B. Thecell/potential inhibitor mixtures are then transported through thevarious branch flow channels into their respective chambers that includethe known receptor ligand. After allowing a suitable time for bindingbetween ligand and receptor, ligand/receptor binding is detected withinthe detection section to identify which, if any, of the potentialinhibitors in fact prevent the ligand from binding to the receptor.

Another example of the usefulness of this configuration is when cellsare contacted with a test agent in the chambers along main flow channelA and a wash cycle is needed before addition of other assay components.This can be facilitated by simply introducing the wash solution intomain flow channel B and transporting the mixture in main flow channel Ato the chambers in main flow channel B where washing occurs.

This configuration can also be arranged such that detection occurs ateach of the chambers as assay solutions are transported along the branchflow channels (A, B, C). This can be useful to establish a baselinesignal in upstream chambers before the final signal is detected atdownstream chambers.

C. Test Compounds

The assay and screening methods described herein can be conducted withessentially any compound. In general terms, the test agent or testcompound is potentially capable of interacting with the component beingassayed (e.g., cell, enzyme, receptor, antibody, cellular organelle). Incellular assays, for example, the component of the cell with which thetest compound potentially interacts can be any molecule, macromolecule,organelle or combination of the foregoing that is located on the surfaceof the cell or located within the cell. For example, if one is screeningfor compounds capable of interacting with certain cellular receptors,the test agents are selected as potentially able to interact with thereceptors of interest (e.g., binding at the binding site of the receptoror affecting binding at the binding site of the receptor, such as anagonist or antagonist). In certain two-hybrid assays (see infra), thetest agent is one that is potentially able to influence the bindinginteraction between the binding proteins of the two fusions (see below).

Consequently, test agents can be of a variety of general typesincluding, but not limited to, polypeptides; carbohydrates such asoligosaccharides and polysaccharides; polynucleotides; lipids orphospholipids; fatty acids; steroids; or amino acid analogs. Further,the compounds can be growth factors, hormones, neurotransmitters andvasodilators, for example. Likewise, the compounds can be of a varietyof chemical types including, but not limited to, heterocyclic compounds,carbocyclic compounds, β-lactams, polycarbamates,oligomeric-N-substituted glycines, benzodiazepines, thiazolidinones andimidizolidinones. Certain test agents are small molecules, includingsynthesized organic compounds.

Test agents can be obtained from libraries, such as natural productlibraries or combinatorial libraries, for example. A number of differenttypes of combinatorial libraries and methods for preparing suchlibraries have been described, including for example, PCT publicationsWO 93/06121, WO 95/12608, WO 95/35503, WO 94/08051 and WO 95/30642, eachof which is incorporated herein by reference.

D. Sample Dilution

The ability to regulate solution flow within the branch channel betweenholding spaces, mixers and chambers allows for sample dilution. Thisfeature of the system is useful in diluting samples. Often drugcandidates in libraries are dissolved in 100% dimethylsulfoxide. Sincethis solvent is incompatible with many biochemical assays until it isdiluted to 1% or less in an aqueous buffer, a multi-stage dilutionprocess can be conducted, whereby different buffer solutions areintroduced into the chambers to achieve the desired level of dilution.

E. Screens with Cells

1. Devices for Use in Cellular Assays

Certain of the microfluidic devices disclosed herein utilize certainelements to facilitate assays conducted with cells, cell components orvarious other types of supports. In general, such microfluidic devicesinclude a main flow channel and one or more branch flow channels influid communication with the main flow channel. The device can include aplurality of branch flow channels so that multiple assays can beconducted at the same time. Typically, the main and branch flow channelsare formed within an elastomeric block.

In addition, a number of elastomeric valves can optionally be disposedalong the length of the main flow channel and positioned adjacent eachof the branch flow lines to regulate solution flow into the differentbranch flow channels. For example, elastomeric valves can be positionedsuch that by selectively activating different valves different solutionsare introduced into different branch flow channels. In this waydifferent cells and/or different reagents can be introduced intodifferent branch flow channels. For instance, cells of the same type canbe introduced into the various branch flow channels but differentreagents subsequently introduced into the different branch flow channelssuch that a variety of different cell assays are conducted in thedifferent branch flow channels. Optional valves can also be locatedwithin the branch flow channels adjacent the point at which the branchflow channel and main flow channel intersect to further control solutionflow through the various channels. The solutions can be transportedthrough the various flow channels by pressurizing or pulling a vacuum onthe various flow channels or under the action of one or more elastomericpumps having the design set forth above that are operatively disposedwith respect to the different flow channels.

The devices can also include a pair of valves operatively disposed withrespect to each of the branch flow channels. The valves can have any ofthe structures described supra. When these valves are closed, theyenclose a volume of solution previously introduced into the branch flowchannel and thus are sometimes referred to herein as “storage valves.”Solution trapped in the space between the valves, as well as any agentswithin the solution (e.g., cells), can then be detected by a detectorlocated to monitor the solution in the space between the two valves.

An example of such an arrangement of storage valves is illustrated ingreater detail in FIG. 10. This figure depicts an elastomeric block1000, in which control channels 1002A, 1002B are disposed with respectto one another such that when the membrane sections 1008A, 1008B thatseparate the control channels 1002A, 1002B from the branch flow channel1006 are extended into the branch channel 1006 a holding space 1010 isformed in which a volume of solution can be retained. An arrangementsuch as this in which two valves substantially block solution flowthrough the holding space is referred to herein as a “pen.” As describedin greater detail below, various other designs utilize valves having agrated-type structure that allows fluid to pass through the holding areawhile retaining cells therein.

One particular exemplary configuration that illustrates certain featuresof microfluidic device that can be utilized to conduct a variety of cellassays is shown in FIG. 19. The device 900 is formed in an elastomericblock and includes an inlet flow channel 912 that is in fluidcommunication with a main flow channel 914, which in turn is in fluidcommunication with a plurality of branch flow channels 916A, 916B and916C. Although FIG. 19 only depicts three branch flow channels, itshould be recognized that many more branch flow channels can also beutilized. The inlet flow channel 912 includes a sample inlet 920 adaptedto allow for the introduction of a sample containing one or more cellsand an additional inlet 932. The inlet flow channel 912 is in fluidcommunication with an additional inlet flow channel 926 that includesinlet 924. The various inlets 924,932 can be utilized to introduce anumber of different solutions and/or agents into the inlet flow channel912. Examples of such solutions, include but are not limited to,buffers, culture medium, dyes for staining cells, substrates of cellularenzymes and ligands for cell receptors. Solutions introduced into inletflow channel 912 via the different inlets can flow into the main flowchannel 914 and into the various branch flow channels 916A, 916B, 916C.Solution flow through the inlet flow channels can be regulated utilizingvalves 918,922,928 and 930.

A plurality of valves 936 and 940 are operatively disposed along mainflow channel 914 and positioned adjacent the branch flow channels 916A,916B to allow solutions to be selectively introduced into the branchflow channels by closing the appropriate valve. The branch flow channels916A, 916B and 916C each include a set of valves 934,938 and 940 asdescribed supra to form a holding space for cells introduced into thebranch flow channels. The branch flow channels 916A, 916B, 916C alsooptionally include valves 944, 946,948.

As indicated supra, each valve in the pair of valves 934,938,940 locatedin the branch flow channels 916A, 916B and 916C can have varying designsto facilitate certain assay methods. For example, as illustrated in thecross section views shown in FIGS. 11A-11C, in some instances themembrane 1122 of the valve 1100 that separates a control channel 1120and a flow channel 1126 include one or more protrusions 1124. Theprotrusions 1124 are of an appropriate size and shape and are spacedrelative to one another such that when the membrane 1122 is extendedinto the flow channel 1126 the protrusions 1124 allow solution to passthrough the space between protrusions while preventing cells to passtherethrough (FIG. 11A). Alternatively, as illustrated in FIG. 11B, theprotrusions 1128 in other valves 1150 are located on a section 1130 ofthe branch flow channel 1126 opposite the membrane 1122 that separates acontrol channel 1120 and the branch flow channel 1126. Here, too, theprotrusions 1128 are designed to allow for passage of a solution betweenthe protrusions while preventing the passage of cells. Still anotheroption is a valve 1180 utilizing both of the foregoing designs (FIG.11C). Thus, the membrane 1122 between the control 1120 and branch flowchannels 1126 includes elastomeric protrusions 1124 as does the section1130 of the branch flow channel opposing the membrane (i.e., protrusions1128). The two sets for protrusions are usually spaced relative to oneanother such that the protrusions at least partially interlock. However,even when interlocked, solution can flow through the protrusions butcells can not. Arrangements using such valves that allow for solutionflow therethrough are referred to herein as “cages.”

In operation, the methods generally involve introducing a solutioncontaining cells into the different branch flow channels. The cellsintroduced in the different branch flow channels can be the same ordifferent. The pair of valves positioned within each of the branch flowchannels are then actuated to form the holding spaces (e.g., pens orcages) to trap or retain cells within the holding spaces. A detector(not shown) is typically positioned to monitor cells within the holdingspaces. Thus, in screening assays involving cells, often the holdingspace is positioned within the detection section. Thus, the holdingspaces can be utilized to hold cells in order to observe celldevelopment and/or to detect signals generated during an assay (e.g.,enzymatic products). This allows one to monitor or detect various cellfeatures (e.g., cytological and toxicology studies) and to determinecell activities (e.g., enzyme activity). Alternatively, substancessecreted from the cells can be detected.

Again referring to FIG. 19, an analysis can be initiated by closingvalve 922 and opening valve 918 to flush sample through sample inlet920. Subsequently, sample can be introduced into the main flow channel914 by closing valve 918 and opening valve 922 such that solutionintroduced via sample inlet 920 flows into main flow channel 914. Valves936, 940 can be utilized to control solution flow into the branch flowchannels 916A, 916B and 916C. For instance, a solution containing cellscan be introduced into all of the branch channels 916A, 916B, 916C byopening all of the valves (e.g., 936 and 940) along the main flowchannel 14, as well as valves 944,946,948 within the branch flowchannels. Alternatively, valves 936,940 within the main flow channel 914and the valves 944,946,948 within the branch flow channels 916A, 916B,916C can be selectively actuated to introduce different solutions, andthus potentially different cells, into the different branch flowchannels 916A, 916B, 916C.

Solutions other than those containing cells, for example, can beintroduced via inlets 924 and 932. Introduction of solution via eitherof these inlets 924,932 can be regulated using valves 928 and 930. Aswith the cell-containing solutions, the same or different solutions canbe introduced into the different branch flow channels 916A, 916B, 916Cusing the valves 936,940 in the main flow channel 914, in combinationwith the valves 944,946,948 in the branch flow channels.

By using valves such as those described above that trap cells within aholding space while allowing for solution flow therethrough (i.e.,cages), one can contact the cells in the holding areas with a variety ofdifferent solutions and agents while still monitoring the cells withinthe holding space. The extent to which the valve is closed can beregulated by controlling the extent of pressure within the controlchannel that actuates the valve.

The branch flow channels can also enable control experiments to beconducted in a facile manner. For instance, a set of test cells can beexamined in one branch flow channel, while a population of control cellsare treated and examined under similar conditions in an adjacent flowchannel.

2. Cells

Essentially any type of cell can be utilized in the microfluidic cellassay devices provided herein. The flow channels utilized in theparticular flow device are sized to accommodate the particular cellsbeing utilized. The cells can be either prokaryotic or eukaryotic. Thecells can also be from any source including, but not limited to,bacteria, yeast, insect, fungal, plant and animal cells. The animalcells can be from mammals or non-mammals. Exemplary mammalian cellsinclude those typically utilized in the art such as CHO, HeLa, HepG2,BaF-3, Schneider, COS, CV-1, HuTu80, NTERA and 293 cells. The cells canbe naturally-occurring cells or can be recombinant cells that harborvectors including an exogenous gene. In some instances, the cellsexpress a cell surface receptor of interest.

In many assays, the cells are alive and are metabolically functioning.The cells can be contained in tissue, blood and cell cultures, forexample. The cells can also be part of cell-containing fluids, includingbut not limited to, spinal fluid, peritoneal fluid, tissue cellsuspensions, samples obtained bone marrow aspirates or lymph nodes suchas from a biopsy. With some applications, certain cell types areseparated from other cells prior to injection into the microfluidicdevice. Such separations can be achieved using any of a variety ofmethods known to those of ordinary skill in the art includingdifferential lysis, differential centrifugation and affinity columns.

Other assays are performed with components from cells. Thus, certainassays are conducted with cell lysates. Still other assays are conductedwith vesicles.

3. Solutions/Agents

A wide variety of solutions can be flowed through the cages that retainthe cells: In general, such solutions include, but are not limited to,culture medium utilized by the cells for growth, wastes generated by thecells and solutions containing various agents that interact orpotentially interact with the cells. The agents can generally includeany substance able to interact with the cell in some way. Certain agentsare potentially able to generate some type of cellular response. Thus,the agents can include, but are not limited to, agents that potentiallybind to a cellular receptor; substrates, cofactors and/or inhibitors ofenzymes; dyes able to selectively label certain cells or cellularcomponents; potential toxicants and the like.

4. Variations

The cages can also be utilized to trap cells and then allow agentssecreted from the cells to pass from the cage and be detecteddownstream. Thus, for example, once cells have been retained in a cage,they can be contacted with various test agents. One way to evaluate theresponse of the cells to the test agent is to monitor agents secretedfrom the cell which pass through the openings in the cage.

The microfluidic devices are also not limited to performing assays withcells. The microfluidic devices can also be used to conduct assays inwhich certain assay components (e.g., a test agent) are attached to sometype of support. A variety of supports can be utilized in the assays,provided the flow channels are sufficiently large to accommodate thesupports. Often the supports are beads manufactured from glass, latex,cross-linked polystyrene or similar polymers (e.g., polyesters and crosslinked polyacrylamide). Other supports are manufactured from gold orother colloidal metal particles. The supports can be of a variety ofshapes, although typically the supports tend to be roughly spherical.

A variety of other supports can be utilized as well depending upon theparticular application. Examples include, but are not limited to,nanoparticles (see, e.g., U.S. Pat. Nos. 5,578,325 and 5,543,158),molecular scaffolds, liposomes (e.g., Deshmuck, D. S. et al. (1990) LifeSci. 28: 239-242; and Aramaki, Y., et al. (1993) Pharm. Res. 10:1228-1231), protein cochleates (stable protein-phospholipid-calciumprecipitates; see e.g., Chen, et al. (1996) J. Contr. Rel. 42: 263-272)and clathrate complexes. Dendrimers can also be utilized in certainapplications and can be synthesized to have precise shapes and sizes andto include a variety of functional groups at the surface to facilitateattachment of various assay agents (see, e.g., Tomalia, D. A. (1990)Angew. Chemie Int. Edn. 29: 138-175).

The supports generally include one or more functional groups for theattachment of various assay components. Exemplary functional groupsinclude hydroxyl, amino, carboxyl and sulfhydryl.

Additional discussion of methods and devices for conducting cell assaysis set forth in copending and commonly owned application entitled“Apparatus and Methods for Conducting Cell Assays,” filed on Apr. 6,2001, and having attorney docket number 020174-003210US, which isincorporated herein by reference in its entirety for all purposes.

V. Combinatorial Synthesis A. Methods

Microfluidic devices having the general arrangement of components asdescribed for high throughput assays, particularly as depicted in FIG.12, can also be utilized to conduct combinatorial orpseudo-combinatorial chemical synthesis. The methods generally parallelthose described supra for the high throughput screening, except thatinstead of assay components and test agents being introduced into theflow channels different reactants are introduced instead. Thus, usingcertain of the devices provided herein, the test agents to be screenedcan be prepared.

Thus, with reference once again to FIG. 12, branch flow channel valves808, 810, 812, 814, 816, 818 are closed and main flow channel valve 806opened. A first reactant is then introduced into the main flow channel802 via inlet 804 and allowed to flow into the chambers 820A-C along themain flow channel. Main flow channel valve 806 is then closed and thebranch flow channel valves 808, 810, 812, 814, 816, 818 opened tointroduce an additional reactant into each of the branch flow channels842A-C. Reactants within each flow channel 842A-C flow into theirchambers 820A-C were they become mixed with the reactant introduced intothe main flow channel 802 to form nascent compounds. The resultingmixture in each chamber can then be transferred into the mixer in linewith the chamber for further mixing/reaction or into a holding space(e.g., in the detection space 828A-C) within the branch flow channelassociated with the chamber.

Additional reagents can be joined to the nascent compounds by variousways. One option is for a single reactant to be introduced into the mainflow channel 802 and the chambers 820A-C located therein. Nascentcompounds in the various flow channels 842A-C are then transported backto the chambers 820A-C for reaction with the newly introduced reactantintroduced via the main flow channel 802. The resulting compounds areagain transferred from the chambers 820A-C to their respective branchflow channel 842A-C. This process can be repeated as many times asnecessary to obtain the final product.

An alternative to this iterative cycle is for additional reactants to beintroduced into the branch flow channels 842A-C and the chambers 820A-Clocated therein. The nascent compounds in each branch flow channel842A-C are then transported back to the chambers 820A-C for reactionwith the newly introduced reactants. Following mixing within thechambers 820A-C, the mixtures are then transferred once again into therespective branch flow chambers 842A-C. This iterative process can berepeated with additional reactants to generate the desired library ofcompounds.

Further guidance regarding combinatorial methods is provide in PCTpublications WO 93/06121, WO 95/12608, WO 95/35503, WO 94/08051 and WO95/30642, each of which is incorporated herein by reference.

B. Compounds

The compounds generated by such methods can be composed of anycomponents that can be joined to one another through chemical bonds in aseries of steps. Thus, the components can be any class of monomer usefulin combinatorial synthesis. Hence, the components, monomers, or buildingblocks (the foregoing terms being used interchangeably herein) caninclude, but are not limited to, amino acids, carbohydrates, lipids,phospholipids, carbamates, sulfones, sulfoxides, esters, nucleosides,heterocyclic molecules, amines, carboxylic acids, aldehydes, ketones,isocyanates, isothiocyanates, thiols, alkyl halides, phenolic molecules,boronic acids, stannanes, alkyl or aryl lithium molecules, Grignardreagents, alkenes, alkynes, dienes and urea derivatives. The type ofcomponents added in the various steps need not be the same at each step,although in some instances the type of components are the same in two ormore of the steps. For example, a synthesis can involve the addition ofdifferent amino acids at each cycle; whereas, other reactions caninclude the addition of amino acids during only one cycle and theaddition of different types of components in other cycles (e.g.,aldehydes or isocyanates).

Given the diversity of components that can be utilized in the methods ofthe invention, the compounds capable of being formed are equallydiverse. Essentially molecules of any type that can be formed inmultiple cycles in which the ultimate compound or product is formed in acomponent-by-component fashion can be synthesized according to themethods of the invention. Examples of compounds that can be synthesizedinclude polypeptides, oligosaccharides, polynucleotide, phospholipids,lipids, benzodiazepines, thiazolidinones and imidizolidinones. As notedabove, the final compounds can be linear, branched, cyclic or assumeother conformations. The compounds can be designed to have potentialbiological activity or non-biological activity.

VI. Variations A. Temperature Controller

With certain assays the ability to regulate temperature is an importantfeature. For example, assays involving denaturation of proteins orthermal cycling reactions during primer extension and nucleic acidamplification reactions require temperature regulation. A number ofdifferent options are available for achieving such regulation that varyin degree of sophistication. Utilizing the following options, one canregulate temperature throughout the device or to selectively regulatethe temperature at particular locations. Furthermore, the temperaturecan be maintained at a relatively constant level or can be controlledaccording to a particular temperature profile or cycle.

One specific approach for regulating temperature within the devices isdisclosed in U.S. Provisional Patent Application entitled “Nucleic AcidAmplification Utilizing Microfluidic Devices,” filed Nov. 16, 2001, andhaving Attorney Docket No. 020174-004420US, the disclosure of which isincorporated herein by reference in its entirety.

Another approach for regulating temperature within the devices is toemploy external temperature control sources. Examples of such sourcesinclude, but are not limited to, heating blocks and water baths. Anotheroption is to utilize a heating element such as a resistive heater thatcan be adjusted to a particular temperature. Such heaters are typicallyutilized when one seeks to simply maintain a particular temperature.Another suitable temperature controller include Peltier controllers(e.g., INB Products thermoelectric module model INB-2-(11-4)-1.5). Thiscontroller is a two-stage device capable of heating to 94° C. Such acontroller can be utilized to achieve effective thermal cycling or tomaintain isothermal incubations at any particular temperature.

In some devices and applications, heat exchangers can also be utilizedin conjunction with one of the temperature control sources to regulatetemperature. Such heat exchangers typically are made from variousthermally conductive materials (e.g., various metals and ceramicmaterials) and are designed to present a relatively large externalsurface area to the adjacent region. Often this is accomplished byincorporating fins, spines, ribs and other related structures into theheat exchanger. Other suitable structures include coils and sinteredstructures. In certain devices, heat exchangers such as these areincorporated into a holding space, chamber or detection regions asdescribed supra. Heat exchangers that can be utilized in certainapplications are discussed, for example, in U.S. Pat. No. 6,171,850.

B. Channel Coatings

In certain methods, the flow channels are coated or treated with variousagents to enhance certain aspects of the assay. For example, dependingupon the nature of the material from which the flow channels are formed,it can be useful to coat the flow channels with an agent that protectsagainst or prevents components of the assay (for example cells,proteins, peptides, substrates, small molecules) from adhering to thewalls of the flow channels or to the sides of the wells through whichthese agents are introduced into the device. One function of thesecoatings is to help ensure the biological integrity of the introducedsample. Another function is to prevent physical interactions betweencells and the walls of the channel that might affect cellular responsesor functions in undesired ways. Examples of suitable coating agentsinclude, but are not limited to, TEFLON, parylene, acrylamides,polyethylene glycol, silanes, and other agents to form self-assembledmonolayers.

Similarly, channels can be modified with a variety of agents to achieveother purposes such as separation and sorting functions, with the goalbeing to prepare the flow channels in accordance with the particularapplication being conducted. More specifically, by properly selectingthe bulk matrix of the flow channel (i.e., the particular choice ofelastomers to utilize in constructing the flow channels), surfacechemistry (i.e., modification of the properties of microchannels createdwithin the elastomer) and the specific modification of regions of theelastomer surface (e.g., by covalent and/or non-covalent attachment ofproteins, peptides, nucleic acids (or their analogs), lipids,carbohydrates) can facilitate the “tuning” of the device to a givenapplication or combination of applications. Methods for modification ofelastomer surfaces include, but are not limited to: (1) copolymerizationwith functional groups during elastomer curing (an example of bulkmodification), (2) oxygen plasma treatment (3) modification ofplasma-treated surfaces with silanizing reagents (e.g.,3-aminopropyltriethoxysilane,N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, dimethylchlorosilane orhexamethyldisilazane) which form self-assembled monolayers on theelastomer surface (which can be used to treat individual flow channels),(4) use of photochemical crosslinking reagents to create patterns ofreactive groups on the elastomer surface (e.g., aryl azide derivativesor quinone-based derivatives), (5) passive modification of the elastomersurface by adsorption.

Adsorption also enables one to create secondary or tertiary layers ofmodification that offer improved properties over primary adsorption. Asa specific example, one can use antibodies against an antigen to createa primary coating of flow channel walls. If antigen is then bound to thebound antibody, one can then create a secondary layer of specificallybound antigen. Antigen bound in, this way can be “presented” to theinterior of the flow channel in a more appropriate way than as apassively adsorbed primary layer. Schemes for creating a plurality oflayers composed of proteins, nucleic acids, lipids or carbohydrates orcombinations thereof will be apparent to the skilled practitioner.

Channels can also be coated with materials that specifically bind toassay components and/or reaction products such as products produced by acell or during an enzymatic assay, for instance. One example of such acoating is one in which the channel is coated with a metal or ametal-derivatized material. Reaction products bearing a metal chelatetag thus become bound to the metal-coated wall or material. Of course, awide variety of other binding pairs could also be utilized assubstitutes for the metal chelating agent and metal. Assays utilizingsuch metal-derivatized materials is discussed in greater detail infra onthe section on enzymatic assays (see also U.S. Pat. No. 6,146,842).

C. Doping Channels with Magnetic Materials

The flow channel elastomeric walls can optionally be doped with magneticmaterials or by integration of a preformed magnet or electromagnet intothe microfluidic device. Examples of magnetic materials that can beincorporated include magnetically polarizable materials such as iron andpermanently magnetized materials. Inclusion of such materials within theflow channel enables magnetic based separations to be performed.External magnets that rotate can in some instances be used to facilitatemixing.

D. Electrodes

The flow channels can also optionally include electrodes to provide anadditional type of control over agent and solution transport.Integration of electrodes into the devices permits electrophoreticseparations or electroosmotic flow to be integrated with pump-driventransport. Suitable electrodes can be formed by sputtering a thin layerof metal (e.g., gold) onto a surface in a flow channel. Othermetallization approaches such as chemical epitaxy, evaporation,electroplating, and electroless plating can also be utilized to form thenecessary conductive material. Physical transfer of a metal layer to thesurface of the elastomer is also available, for example by evaporating ametal onto a flat substrate to which it adheres poorly, and then placingthe elastomer onto the metal and peeling the metal off of the substrate.A conductive electrode can also be prepared by depositing carbon black(e.g., Cabot Vulcan XC72R) on the elastomer surface, either by wiping onthe dry powder or by exposing the elastomer to a suspension of carbonblack in a solvent which causes swelling of the elastomer, (such as achlorinated solvent in the case of PDMS).

E. Membrane Integration

1. General

An additional element or module that can be incorporated into themicrofluidic devices disclosed herein are elastomeric structures thatinclude semi-permeable membranes that allow certain agents to passtherethrough, while other agents are not. As described in greater detailbelow, such modules can be used to perform a variety of usefulfunctions. Such functions include, but are not limited to, dialysis toremove unwanted agents, purification and concentration.

2. Membrane Composition

The membranes utilized in the devices can be formed from almost anycommercially-available polymer. Suitable commercially-available polymermembranes include, but are not limited to, cellulose, various celluloseesters (in particular cellulose acetate), nitrocellulose, polycarbonate,polyethylene, nylon, polypropylene, polysulfone, polyethersulfone,polystyrene, Teflon (PTFE), polyvinylchloride (PVC) andpolyvinylidenedifloride (PVDF). Membranes can also be formed from porousinorganic materials, including glass, quartz, and anodically-treatedalumina (Al203) or other oxides, for example.

3. Preparation

Two basic methods exist for integrating membranes in elastomer-basedmicrofluidic devices: multilayer bonding-based methods andencapsulation-based methods. These methods correspond to multilayer softlithography (MSL) and sacrificial-layer encapsulation (SLE) methods;these methods are described in detail in PCT Application No. 00/17740,in U.S. application Ser. No. 09/605,520, filed Jun. 27, 2000, and inU.S. application Ser. No. 09/724,784, filed Nov. 28, 2000, each of whichis incorporated by reference in its entirety for all purposes. Ingeneral, multilayer soft lithography fabricates structures by moldingand curing of polymers (elastomers) on micromachined substrates; eachlayer is cured separately, and then the layers are assembled togetherand bonded. The chemistry of the layers is chosen and/or manipulated toallow bonding of the cured layers. In the sacrificial layerencapsulation scheme, devices are fabricated by sequentially addinglayers of elastomer and curing them, with microfluidic channels definedby patterned sacrificial layers added between layers of elastomer. Thismethod requires care to choose a sacrificial layer compatible with thepolymer chosen, but has the advantage that uncured polymer generallyforms a good bond to cured polymer even without specific manipulation ofthe chemistry of the layers.

Different elastomeric layers can be joined using different approaches.In some instances, elastomeric layers are bonded together chemically,using chemistry that is intrinsic to the polymers comprising thepatterned elastomer layers. For example, sometimes bonding isaccomplished utilizing two component “addition cure” bonding.

In certain approaches, the various layers of elastomer are boundtogether by a heterogenous bonding in which the layers have a differentchemistry. Alternatively, homogenous bonding can be used in which alllayers are of the same chemistry. Thirdly, the respective elastomerlayers can optionally be glued together by an adhesive. Yet anotheroption if the elastomeric layers are thermoset elastomers is for thelayers to be bonded together by heating.

With certain homogeneous bonding approaches, the elastomeric layers arecomposed of the same elastomer material, with the same chemical entityin one layer reacting with the same chemical entity in the other layerto bond the layers together. In some instances, bonding between polymerchains of like elastomer layers can result from activation of acrosslinking agent due to light, heat, or chemical reaction with aseparate chemical species.

In certain heterogenous approaches in contrast, the elastomeric layersare composed of different elastomeric materials, with a first chemicalentity in one layer reacting with a second chemical entity in anotherlayer. For example, in certain heterogenous approaches, the bondingprocess used to bind respective elastomeric layers together involvesbonding together two layers of RTV 615 silicone. RTV 615 silicone is atwo-part addition-cure silicone rubber. Part A contains vinyl groups andcatalyst; part B contains silicon hydride (Si—H) groups. Theconventional ratio for RTV 615 is 10A:1B. For bonding, one layer can bemade with 30A:1B (i.e., excess vinyl groups) and the other with 3A:1B(i.e., excess Si—H groups). Each layer is cured separately. When the twolayers are brought into contact and heated at elevated temperature, theybond irreversibly forming a monolithic elastomeric substrate.

Alternatively, other bonding methods can be used, including activatingthe elastomer surface, for example by plasma exposure, such that theelastomer layers/substrate bond when placed in contact. For example, oneapproach to bonding elastomer layers together that are composed of thesame material is set forth by Duffy et al. (1998) Analytical Chemistry70:4974-4984, incorporated herein by reference in its entirety. Such anapproach involves exposing polydimethylsiloxane (PDMS) layers to oxygenplasma to cause oxidation of the surface, with irreversible bondingoccurring when the two oxidized layers are placed into contact.

Yet another approach to bonding together successive layers of elastomeris to utilize the adhesive properties of uncured elastomer.Specifically, a thin layer of uncured elastomer such as RTV 615 isapplied on top of a first cured elastomeric layer. Next, a second curedelastomeric layer is placed on top of the uncured elastomeric layer. Thethin middle layer of uncured elastomer is then cured to produce amonolithic elastomeric structure. Alternatively, uncured elastomer canbe applied to the bottom of a first cured elastomer layer, with thefirst cured elastomer layer placed on top of a second cured elastomerlayer. Curing the middle thin elastomer layer again results in formationof a monolithic elastomeric structure.

Where encapsulation of sacrificial layers is employed to fabricate theelastomer structure, bonding of successive elastomeric layers can beaccomplished by pouring uncured elastomer over a previously curedelastomeric layer and any sacrificial material patterned thereupon.Bonding between elastomer layers occurs due to interpenetration andreaction of the polymer chains of an uncured elastomer layer with thepolymer chains of a cured elastomer layer. Subsequent curing of theelastomeric layer creates a bond between the elastomeric layers andresults in a monolithic elastomeric structure.

Although materials used for membranes typically are not elastomers, themethods used for fabrication of structures with integrated membranes aresimilar. Thus, for example, and as illustrated in the cross-sectionalview shown in FIG. 14, certain multilayer soft lithography methodsinvolve preparing two layers of elastomer 1406, 1408 by softlithographic techniques in which elastomer is spin coated onto molds1402, 1404. Membrane 1410 is bonded to one of the elastomer layers 1408after elastomer layer 1408 has been peeled off of mold 1404 and cured.Elastomer layer 1406 is then turned over and assembled together with theassembly comprising elastomer layer 1408 and membrane 1410, all threeelements being bonded together chemically. The chemistry of theelastomer layers 1406, 1408 and membrane 1410 to be chosen to allowbonding of the cured elastomer layers to the membrane. This can beaccomplished by either using elastomers compatible with the membranechemistry or by modifying the chemical groups on the surface of themembrane to allow bonding to the elastomer in use.

An example of the first process is to use a photocurable elastomer(e.g., Ebecryl 270, a urethane available from UCB Chemical) and bond themembrane 1410 to the elastomer 1408 using a photoinitiated reaction. Anexample of the second process involves modification of a cellulosemembrane 1410 to produce a surface reactive towards hydroxyl groups,which groups are present at the surface of a polyurethane elastomerproduced with an excess of the diol component. Techniques formodification of polymer surfaces to allow the attachment of moleculesare well known in the art, as these are the basis of solid-phasesynthesis (see, e.g., Hermanson, G. T., et al. (1992) “ImmobilizedAffinity Ligand Techniques” Academic Press, San Diego; and Zaragoza, D.F. (2000) “Organic Synthesis on Solid Phase Supports, Linkers,Reactions,” Wiley-VCH, New York). The simplest rendition of membranedevices utilizing MSL techniques involves assembly of both elastomerlayers 1406, 1408 with the membrane 1410 such that microfluidic channelsface the membrane. This is somewhat different from other MSL schemes inwhich general layers are assembled “top-to-bottom” (i.e., with channelson the bottom side of each layer) rather than face-to-face. Thetechnique and intent are the same, however.

An example of an encapsulation scheme is illustrated in FIG. 15. Thisexample shows an assembly including a first elastomer layer 1504attached to membrane 1506 and overlayed with a second elastomer layer1502. As shown in this figure, some of the sacrificial layers 1508 arepatterned directly on the membrane 1506, the elastomer 1504 coated ontop of the sacrificial layer 1508 and cured. Multiple layers ofelastomer and sacrificial layer can be added in order to make activeelastomeric fluidic devices. The membrane-containing assembly is thenturned over, a sacrificial layer 1508 patterned on the other side, andan elastomer layer 1510 built on that side as well. Again, multiplelayers can be added. Finally, the sacrificial material is removed,leaving microchannels on both sides of the membrane in fluidcommunication with one another through the membrane. The advantage ofthis scheme is that uncured elastomer is very likely to form a chemicalbond with the polymer membrane. Even with substrates that form nochemical bond with the elastomer, physical impregnation of the elastomerinto the membrane matrix can yield a very strong attachment by thismethod.

Another example of a device having a membrane component is illustratedin FIG. 16. As can be seen, this particular device 1600 also includes aflow channel 1604 in one layer of elastomer 1602 and a second flowchannel 1608 in a second elastomer layer 1610. The two flow channels1604, 1608 are separated by a membrane 1606. In this device 1600,however, the two flow channels 1604, 1608 are not oriented parallel toone another, but instead cross at an angle. The upper flow channelincludes a sample input 1610 and an outlet 1612.

For inert membranes for which no elastomer can be found to form achemical bond (e.g., Teflon), practical devices can nonetheless beassembled. Assembly follows the MSL scheme described supra, but the bondbetween the layers is. limited to that provided by nonspecific (e.g.,Van der Waals) forces. As elastomer microfluidic chips form a hermeticseal with nearly any flat surface, this seal usually is sufficientlystrong to allow operation of the device.

4. Exemplary Uses

In general, the devices including a membrane that separates two flowchannels can be used in a variety of separation and concentrationapplications. As illustrated in FIGS. 17A-C. for example, the membranedevices or modules can be utilized to perform a number of differentfunctions. FIG. 17A, shows a device in which two parallel channels 1702,1706 are separated by a membrane 1704, with a flow of solution in onechannel and a slower flow of solution or static fluid in the otherchannel (solution flow represented by arrows). Such an arrangement isuseful for removing small molecules from a mixture. For example, thistype of an arrangement can be used in dialysis. A specific applicationof such a device, is to remove salt from a biological sample (e.g., saltfrom an enzyme preparation).

FIG. 17B depicts another arrangement in which parallel channels 1708,1710 (or chambers) are separated by a membrane 1704. In this instance,applying pressure to one channel (e.g., channel 1708) causes solution toflow through the membrane 1704, with particles/molecules larger than acertain size retained on one side (e.g., in channel 1708) andparticles/molecules smaller than that size passing through into channel1710. Such an arrangement can be considered as an ultrafiltrationdevice. This device is useful for removing small particles/moleculesfrom a sample and retaining the larger molecules. The applied pressureallows larger molecules to be concentrated. Typically, the appliedpressure is some type of gas (e.g., air).

FIG. 17C shows another configuration in which parallel channels 1712,1714 (or chambers) are separated by a membrane 1704, with a plurality ofinlets 1716 a, 1716 b, 1716 c available to flow in a sample and otheroptional agents. In this particular device, the membrane compositionallows binding of a component of the sample to the membrane 1704.Another solution can be flowed into channel 1712 via inlet 1716 b towash away the non-bound components of the mixture; yet another solutioncan be introduced into channel 1712 (e.g., via inlet 1716 c) to releasethe bound component from the mixture. Released components can bedirected to an outlet 1718 in channel 1712 or on the opposite side ofthe membrane 1704 via outlet 1720.

In some assays, cells are bound to the membrane 1704 (or anywhere alongflow channel 1712) and an agent that affects the cell is added into flowchannel 1704 via one of the inlets 1716 a-c. Small molecular weightcompounds produced by the cells pass through the membrane 1704 and arecollected through outlet 1720 for further analysis.

F. Separation Module

A separation module or modules can also be incorporated into the presentmicrofluidic devices. Such a module allows components within a solutionto be separated, or at least partially separated, from one another. Oneparticular module is illustrated in FIGS. 18A and 18B. In general themodule 1800 includes a flow channel 1802, which is in fluidcommunication with an inlet 1806 for introducing separation material anda waste outlet 1808. The region in which separation material is locatedis referred to as the column region 1804. Various control valves 1810,1812, 1814, 1816 can be actuated to regulate introduction of separationmaterial, the exit of waste and flow through the column 1804.

Separation material is packed along the column region 1804 by openingthe valve 1810 in the separation material input channel 1818 and thevalve 1816 in the waste channel 1820. The column separation material isallowed to flow from the separation material input channel 1818 to thewaste collection site 1808. After the column has been packed, theseparation material input channel 1818 and the waste channel 1820 areclosed using valves 1810 and 1816, respectively. If the separationmodule is part of a device such as shown in FIG. 12 or 13, the solutionflowing into the column 1804 typically is a solution moving downstreamfrom an upstream location in which various assay components have beenadded (and often mixed). The separation column 1804 can be used toseparate components in an assay solution mixture prior to detection in adetection section. This module can be used as a stand alone device,however, with a separate sample inlet (not shown) in fluid communicationwith a section of the flow channel 1802 upstream of the column region1804.

A wide variety of separation materials can be utilized in the column toeffect separation. In general, the separation material can include anymaterial that allows separation of components according to affinity,size, mobility, and the like. Specific examples of separation materialinclude, but are not limited to, size exclusion material, ion exchangematerial, cross-linked polymeric gels (e.g., polyacrylamide) andaffinity chromatography material.

Size exclusion materials can be used to separate components based uponsize. Such separations can be conducted as part of a desalting operationor to separate similar compounds that differ in size (e.g., proteins ofdifferent molecular weight). Ion exchange chromatographic material canbe utilized to separate charged agents and/or to exchange one counterionfor another. Affinity chromatography materials can be used toselectively bind certain components.

Certain separation modules are designed to conduct electrophoreticseparations. Thus, the column 1804 includes a gel matrix (e.g.,polyacrylamide or agarose). Electrodes of the type described supra canpositioned at opposing ends of the column and used to apply a voltageacross the column 1804.

The amount of pressure required to flow the sample fluid through thecolumn can cause separation of joints within the microfluidic device. Toprevent separation of layers or joints, an assembly 1850 includes amicrofluidic device 1854 sandwiched between two metal plates 1852 and1856. A pressure screw 1858 can be utilized to apply pressure betweenthe two plates 1856 and 1852 to keep the RTV layers of the microfluidicdevice from separating at high pressure.

Other types of separation systems are described in U.S. provisionalpatent application entitled “Microfluidic Sample Separation Device,”having attorney docket number 020174-004100US, filed on Apr. 6, 2001,which is incorporated by reference herein in its entirety for allpurposes.

VII. Exemplary Applications

The microfluidic devices disclosed herein can be utilized to conduct avariety of different assays. Essentially any biological assay or libraryscreening application can be performed with the microfluidic devicesthat are described herein, provided none of the components of the assayor screen are incompatible with the size of the microfluidic channels.For example, the present high throughput screening devices can be usedto screen for any agents that affect the activity of any class of“druggable” targets (i.e., a target that is able to be modulated by asmall molecule to produce a desired phenotypic change in cell targets).Potential druggable targets include, but are not limited to, G-proteincoupled receptors (GPCRs), cytokines and cytokine receptors, nuclearreceptors (ligand-dependent transcription factors), signaling processes(e.g., receptor-ligand interactions, calcium mobilization, kinases andphosphatases, second messengers and transcription factors), proteases,ion channels, and determinants of cytotoxicity (e.g., pro- andanti-apoptotic processes and cell death). These targets can be addressedby the various types of assays described herein, including, for example,fluorescent detection technologies such as fluorescence intensitydeterminations, fluorescence polarization, fluorescence resonance energytransfer, time-resolved techniques and fluorescence correlationspectroscopy.

The following include a non-exhaustive list of illustrative assays thatcan be conducted with the microfluidic devices provided herein, andillustrate the nature of the targets that can be investigated and thetypes of detection schemes that can be utilized.

A. Enrichment of Selected Cells/Cellular Components

1. General

Certain cells and cell components can be selectively enriched within asection of a flow channel. The term “cell component” broadly refers toan agent that is part of the cell, contained within the cell or producedby the cell. Thus, the component can be a structural agent (e.g., amembrane, tubule or protein) a cytoplasmic component or a productgenerated through a metabolic or catabolic activity of the cell, forinstance. The “enrichment” section or zone can be located within adetection section or at other sections of the flow channel system, forexample. An enrichment section in general includes an agent that is amember of a binding pair and that selectively interacts with aparticular target cell or cellular component of interest that includesthe other member of the binding pair. In the case of cells, the agentcan be a ligand that interacts with an antiligand (e.g., a receptor) onthe cell surface. The interaction between the agent and the selectedcells or cell products can be a stable interaction in which the cell orcell component becomes immobilized within the enrichment section or aninteraction that simply slows flow of the selected cells or cellproducts through the enrichment region relative to other cells or cellproducts.

The enrichment section can be designed to contain agents thatspecifically interact with a wide variety of molecules on the surface ofthe target cell or that are part of the cell component. Examples ofsuitable agents that can function as one member of the binding pairinclude, but are not limited to, lectins, enzyme cofactors, enzymeinhibitors, ligands for receptors and antibodies that recognizeparticular cell markers. Such antibodies can be directed toward any of avariety of different markers displayed on the target cell surface.Examples of such markers or antigens include markers for T cells or Tcell subsets, B cells, monocytes, leukocytes, myeloid cells, HLA ClassII positive cells and stem cells.

2. Coated Channels

One option for presenting the agent to the selected cells is to coat theenrichment section with the agent as described supra in the channelcoating section. Thus, for example, a ligand can be attached to theinterior surface of the flow channel that binds to an antiligand exposedon the exterior surface of the selected cells. Alternatively, multiplelayers can be formed on the flow channel surface. For instance, anantibody that specifically binds to a particular antigen that isrecognized by the target cells of interest can be coated onto the flowchannel surface. The antigen can then be added as a second layer bycontacting the immobilized antibody with the antigen. Thus, the flowchannel displays antigen to cells passing through the enrichment region.Cells that bind the antigen (e.g., cells expressing a receptor thatbinds the antigen) are thus retained within the enrichment zone whileother cells can flow through.

3. Coated Supports

Another option for preparing an enrichment region is to utilize supportsthat are coated with the binding pair member such that target cells orcell components having the other binding pair member become complexedwith the coated supports. Binding pair members can be attached tosupports utilizing a number of well established chemistries. Forexample, tosylactivated forms of the supports can be utilized to customcoat the supports with the agent of choice. Streptavidin coated supportscan be conjugated with biotinylated agents.

Supports can be retained within a particular desired region in a numberof different ways. For instance, porous class frits or plugs of porousmaterials (e.g., polymeric gels such as agarose gel) can be utilized atthe inlet and outlet of the enrichment section. If paramagnetic supportsare utilized, coated supports can be retained utilizing applied magneticfields.

4. Impregnated or Coated Membranes

The enrichment section can also utilize coated or impregnated membranesto selectively enrich particular cells or cell components. Thesemembranes are either coated or impregnated with an agent/binding pairmember such as those described supra. The membrane can be utilized toselectively retain certain targets while allowing other molecules topass through the membrane and from the enrichment region for transportto another section of the microfluidic device (e.g., a waste outlet).Membranes of this type can be fabricated into the devices describedherein as set forth in the membrane integration section supra.

Using such membranes, one can selectively retain molecules above acertain cutoff size (i.e., macromolecules or cells that are larger thanthe pore sizes within the membrane) and/or retain target cellcomponents. Thus, for example, by utilizing a an appropriate membrane,one can retain cells within the enrichment section by virtue of theirsize (i.e., they are too large to pass through the membrane), whilesimultaneously retaining a cell product produced by the cell by virtueof its binding to its binding partner that is impregnated into or coatedon the membrane. In this way, one can enhance the ability to detect cellproducts that are formed at very low levels. A variety of membranes thatcan be utilized in selective enrichment of certain agents is discussed,for example, by Tomlinson, et al. (1995) J. Cap. Elect. 2: 97-104; andTomlinson, et al. (1995) J. High Res. Chromatogr. 18: 381-3.

5. Variations

Of course, the enrichment sections can also be utilized to enrich foragents other than cells or cell components. The detection sections canalso or alternatively be designed to enrich any number of other agents.Thus, the enrichment section can be utilized to enrich for a particularassay reagent (e.g., an enzyme substrate) or agent that reacts with aproduct generated by a cell, for example. Those having ordinary skill inthe art will appreciate that a large number of other agents can also beenriched utilizing the devices and according to the methods disclosedherein.

While the above methods have described systems in which one member of abinding pair is utilized to selectively retain target cells or cellcomponents within the enrichment section, the reverse approach can alsobe utilized in which a binding pair member that is present on cellsother than the target cell or cell component is used to retainnon-target cells while the target cell or cell component pass throughthe enrichment section. The target cells can subsequently be collectedand assayed free of other cells.

B. Detecting Presence of Particular Cells

The enrichment methods just described provide some techniques fordetecting selected target cells of interest. Various other methods arealso available for detecting the presence of target cells. One option isto provide a detection section which permits one to examine cellmorphology either directly or more typically by conventional microscopy.Thus, a microscope can be aligned with a section of the microfluidicdevice in which the target cells are contained (e.g., within a cell penor cage as described supra or within an enrichment section).Identification of some cells can in some instances be enhanced byapplying histological stains that are known in the art to selectivelystain the cells of interest.

As discussed above, another strategy is to detect target cells accordingto particular protein markers that are expressed by the cells ofinterest. Such markers are typically identified using labeled antibodiesthat specifically recognize the distinctive cell marker.

Yet another option is to examine the mRNA (or a nucleic acid moleculederived therefrom such as a cDNA) that is transcribed by cells, asdifferent cell types typically have different mRNA profiles. This isparticularly true of certain types of cells. Examples of such cellsinclude, but are not limited to, cells infected with bacteria orviruses, cancerous cells which transcribe specific mRNAs, cellstranscribing mRNA isoforms that include one or more sites of mutation,keratinocytes (keratin mRNA), and chondrocytes (aggrecan mRNA). Oneapproach for utilizing this approach involves using primer pairs thatenable one to distinguish between mRNA and genomic DNA signal. Forexample, one can select primers that amplify a short segment of mRNAwhere an intron is present in the corresponding genomic sequence.

Certain assays involve isolating cells within a region of themicrofluidic device, such as in a pen or cage as described supra. Anassay mixture containing a cell lysing agent or detergent (e.g.,TWEEN-20), optionally an RNase inhibitor to inhibit the degradation ofthe mRNA and the agents necessary to conduct a RT-PCR reaction (e.g.,primers and polymerase) are introduced into the microfluidic device andtransported to the cells. By utilizing heaters such as described above,one can conduct the cycling steps to perform the RT-PCR. Amplificationproducts can be detected by utilizing labeled primers which thusgenerate labeled products. In some assays, amplified product canoptionally be separated from labeled primer in a separation module asdescribed above.

C. Cell Reporter Assays

A number of different cell reporter assays can be conducted with theprovided microfluidic devices. One common type of reporter assay thatcan be conducted include those designed to identify agents that can bindto a cellular receptor and trigger the activation of an intracellularsignal or signal cascade that activates transcription of a reporterconstruct. Such assays are useful for identifying compounds that canactivate expression of a gene of interest. Two-hybrid assays are anothermajor group of cell reporter assays that can be performed with thedevices. The two-hybrid assays are useful for investigating bindinginteractions between proteins. These are discussed in greater detail inthe following sections.

Often cell reporter assays are utilized to screen libraries ofcompounds. Although a variety of microfluidic devices including thevarious components disclosed herein can be used, a device such as thatshown in FIG. 9 can be utilized in the high throughput screening of alibrary of compounds for their ability to activate a receptor orinfluence binding between binding proteins. In general such methodsinvolve introducing the cells into the main flow channel so that cellsare retained in the chambers located at the intersection between themain flow channel and branch channels. Different test agents (e.g., froma library) can then be introduced into the different branch channelswhere they become mixed with the cells in the chambers.

Alternatively, cells can be introduced via the main flow channel andthen transferred into the branch channel, where the cells are stored inthe holding areas. Meanwhile, different test compounds are introducedinto the different branch flow channels, usually to at least partiallyfill the chambers located at the intersection of the main and branchflow channels. The cells retained in the holding area can be released byopening the appropriate valves and the cells transferred to the chambersfor interaction with the different test compounds. Once the cells andtest compounds have been mixed, the resulting solution is returned tothe holding space or transported to the detection section for detectionof reporter expression. The cells and test agents can optionally befurther mixed and incubated using mixers of the design set forth above.

1. Receptor Binding and Gene Activation

The cells utilized in screening compounds to identify those able totrigger gene expression typically express a receptor of interest andharbor a heterologous reporter construct. The receptor is one whichactivates transcription of a gene upon binding of a ligand to thereceptor. The reporter construct is usually a vector that includes atranscriptional control element and a reporter gene operably linkedthereto. The transcriptional control element is a genetic element thatis responsive to an intracellular signal (e.g., a transcription factor)generated upon binding of a ligand to the receptor under investigation.The reporter gene encodes a detectable transcriptional or translationalproduct. Often the reporter (e.g., an enzyme) can generate an opticalsignal that can be detected by a detector associated with a microfluidicdevice. General cell reporter systems are discussed in U.S. Pat. No.5,436,128. In addition to their use in identifying compounds that areligands of receptors that trigger gene activation, the methods can alsobe used to identify compounds that are agonists or antagonists ofreceptors of interest.

A wide variety of receptor types can be screened. The receptors oftenare cell-surface receptors, but intracellular receptors can also beinvestigated provided the test compounds being screened are able toenter into the cell. Examples of receptors that can be investigatedinclude, but are not limited to, ion channels (e.g., calcium, sodium,potassium channels), voltage-gated ion channels, ligand-gated ionchannels (e.g., acetyl choline receptors, and GABA (gamma-aminobutyricacid) receptors), growth factor receptors, muscarinic receptors,glutamate receptors, adrenergic receptors, dopamine receptors (see,e.g., U.S. Pat. Nos. 5,401,629 and 5,436,128). Additional receptorsinclude the G-protein coupled receptors, specific examples of whichinclude substance K receptor, the angiotensin receptor, the a- andp-adrenergic receptors, the serotonin receptors, and PAF receptor (see,e.g., Gilman (1987) Ann. Rev. Biochem. 56: 625-649).

A number of different reporters can be used to detect gene activation.Certain reporter gene encode proteins that are inherently detectable.One specific example of such a reporter is green fluorescent protein.Fluorescence generated from this protein can be detected with variouscommercially-available fluorescent detection systems. Other reporterscan be detected by staining. Most commonly, however, the reporter is anenzyme that generates a detectable signal when contacted with anappropriate substrate. Often the reporter is an enzyme that catalyzesthe formation of a detectable product. Suitable enzymes include, but arenot limited to, proteases, nucleases, lipases, phosphatases andhydrolases. Typically, the reporter encodes an enzyme whose substratesare substantially impermeable to eukaryotic plasma membranes, thusmaking it possible to tightly control signal formation. Specificexamples of suitable reporter genes that encode enzymes include, but arenot limited to, CAT (chloramphenicol acetyl transferase; Alton andVapnek (1979) Nature 282: 864-869); luciferase (lux); β-galactosidase;LacZ; β-glucuronidase; and alkaline phosphatase (Toh, et al. (1980) Eur.J. Biochem. 182: 231-238; and Hall et al. (1983) J. Mol. Appl. Gen. 2:101), each of which are incorporated by reference herein in itsentirety. Other suitable reporters include those that encode for aparticular epitope that can be detected with a labeled antibody thatspecifically recognizes the epitope.

The cells utilized in the cell based assay can include any of thosedescribed supra. In general, the cells must express the receptor ofinterest. The receptor can be an endogenous receptor or a receptorexpressed from a heterologous construct that includes the gene thatencodes for the receptor of interest. In the latter instance, the cellmust be able to transfected with the construct and able to express theheterologous receptor.

2. Two Hybrid Assays

Another general category of cell assays that can be performed is the twohybrid assays. In general, the two-hybrid assays exploit the fact thatmany eukaryotic transcription factors include a distinct DNA-bindingdomain and a distinct transcriptional activation domain to detectinteractions between two different hybrid or fusion proteins. Thus, thecells utilized in two-hybrid assays include the construct (s) thatencode for the two fusion proteins. These two domains are fused toseparate binding proteins potentially capable of interacting with oneanother under certain conditions. The cells utilized in conductingtwo-hybrid assays contain a reporter gene whose expression depends uponeither an interaction, or lack of interaction, between the two fusionproteins.

In standard two-hybrid systems, interaction between the two bindingproteins results in expression of the reporter gene (see, e.g., Fields,S, and Song, O. (1989) Nature 340: 245; Bartel, P. L., and Fields, S.(1995) Methods in Enzymology 254: 241-263; Heery, D. M., et al. (1997)Nature 387: 733-736; and Bartel, et al. (1993) Biotechniques 14: 920).Reverse hybrid systems, in contrast, are designed such that interactionbetween the two binding proteins suppresses reporter expression. Inthese systems, reporter expression is triggered when a compound inhibitsthe interaction between the two binding proteins (see, e.g., PCTpublication WO 95/26400). Two-hybrid assays can be utilized to establishinteractions between two known proteins or to search a genomic or cDNAlibrary for proteins that interact with a target protein.

D. Binding Assays

1. General

A wide variety of binding assays can be conducted utilizing themicrofluidic devices disclosed herein. Interactions between essentiallyany ligand and antiligand can be detected. Examples of ligand/antiligandbinding interactions that can be investigated include, but are notlimited to, enzyme/ligand interactions (e.g., substrates, cofactors,inhibitors); receptor/ligand; antigen/antibody; protein/protein(homophilic/heterophilic interactions); protein/nucleic; DNA/DNA; andDNA/RNA. Thus, the assays can be used to identify agonists andantagonists to receptors of interest, to identify ligands able to bindreceptors and trigger an intracellular signal cascade, and to identifycomplementary nucleic acids, for example. Assays can be conducted indirect binding formats in which a ligand and putative antiligand arecontacted with one another or in competitive binding formats well knownto those of ordinary skill in the art.

Because the microfluidic devices typically include a plurality of branchflow channels and holding spaces that allow multiple analyses to beconducted at the same time, a large number of assays can be conducted ina short period. Throughput can be increased even further by utilizingadditional multiplexing techniques. In this way, different ligands orantiligands can be attached to different supports and a plurality ofsupports assayed within a single branch flow channel. Active ligands orantiligands can be identified on the basis of the distinguishablesupports. For example, assays can be conducted using supports that canbe distinguished by physical, chemical, visual or other means. Morespecifically, supports can be distinguished from one another on thebasis of different composition, size, color, shape, magnetic properties,chemical properties, electronic properties, fluorescent emission, forexample. Specific examples of supports that can be distinguished on thebasis of different fluorescent emissions are Luminex beads (LuminexCorporation) and Quantum dots (Quantum Dot Corporation). Sorting and/orquantitation is based upon support size, wavelength and/or amount ofsignal generated (e.g., fluorescence). Binding assays generally involvecontacting a solution containing ligands with a solution containingantiligands and allowing the solutions to remain in contact for asufficient period such that binding partners form complexes. The ligandand/or antiligand is usually labeled. Any of a variety of differentlabels can be utilized as described above. Ligands and antiligands canbe contacted within the main or branch flow channels. More typically,however, contact occurs within the chambers and/or holding spaces (e.g.,pens or cages) described supra. Solutions containing the ligands andantiligands can be mixed and/or incubated by pumping solutions back andforth between chambers and holding spaces and/or by using the mixersdescribed supra. Complexes typically are detected within a detectionsections along the flow channel. The detection section can includeholding spaces (e.g., pens or cages) as described supra. The type ofdetector and detection method utilized depends upon the type of labelused to label the ligand or antiligand.

2. Heterogeneous Formats

Heterogenous binding assays involve a step in which complexes areseparated from unreacted agents so that labeled complexes can bedistinguished from uncomplexed labeled reactants. Often is achieved byattaching either the ligand or antiligand to a support. After ligandsand antiligands have been brought into contact, uncomplexed reactantsare washed away and the remaining complexes subsequently detected.

The heterogeneous assays performed with the microfluidic devicesdisclosed herein generally involve contacting a solution containing aligand and a solution containing an antiligand with one another underconditions and for a sufficient period of time to allow aligand/antiligand complex to form. Since the ligand or antiligand islabeled, any complexes formed can be detected on the basis of the labelin the complex.

The assays can be conducted in a variety of ways. One approach involvesanchoring an antiligand of interest to some type of a solid support andcontacting the antiligand with a solution containing ligands. Labeledligands that do not form complexes are washed away under conditions suchthat complexes that are formed remain immobilized to the solid support.The detection of complexes immobilized to the support can beaccomplished in a number of ways. If the non-immobilized ligand islabeled, the detection of label immobilized on the solid supportindicates that a ligand/antiligand complex has been formed. If, however,the non-immobilized ligand is not labeled, complexes can nonetheless bedetected by indirect means. For instance, a labeled antibody thatspecifically binds to the ligand can be utilized to detect complexesanchored to the support.

Alternatively, ligands and antiligands can be contacted in solution.Complexes can then be separated from uncomplexed ligands and antiligandsand complexes detected. One approach for conducting such an assay is tocontact an antiligand of interest with a test solution potentiallycontaining a ligand that binds to the antiligand. The resulting mixturecan then be contacted with an immobilized antibody that specificallybinds to the antiligand to immobilize any complexes that have beenformed. Labeled antibodies specific for the ligand can then be contactedwith any immobilized complexes to detect the presence of such complexes.

A variety of strategies are available for immobilizing complexes. Onegeneral approach is to use some type of support that can pass throughthe flow channels. The support is used to bear the ligand or antiligandduring a test. These mobile supports can be immobilized within the cagesdescribed supra. The size of the cages is such that the supports areretained within the cage but different solutions can be passed throughthe limited openings in the cage.

Another immobilization option is to attach a ligand or antiligand (i.e.,one member of a binding pair) to the surface of a flow channel. Theligand (antiligand) attached to the flow channel can be attacheddirectly to the flow channel surface or via a linker. In general, theflow channel surface and the ligand (antiligand) being attached to thechannel surface need appropriate chemical functionality such that thefunctional groups borne by these two entities can react with one anotherand become attached. Often the attachment is achieved by formation of acovalent bond between the binding pair member and surface, althoughelectrostatic, hydrogen bond interactions and hydrophobic interactionscan also act to attach the binding pair member and surface.

A variety of linkers can be utilized to attach the ligand (antiligand)to the flow channel wall. A variety of linkers can be utilized. Thelinkers typically are bifunctional, with a functional group at one endable to react with a functional group on the channel surface and afunctional group on the other end able to react with a functional groupborne by the ligand (antiligand) to be attached to the flow channelsurface. The functional groups at each end of such linkers can be thesame or different. Examples of suitable linkers include straight orbranched-chain carbon linkers, heterocyclic linkers and peptide linkers.Exemplary linkers that can be employed are available from PierceChemical Company in Rockford, Ill. and are described in EPA 188,256;U.S. Pat. Nos. 4,671,958; 4,659,839; 4,414,148; 4,669,784; 4,680,338;4,569,789 and 4,589,071, Eggenweiler, H. M, Drug Discovery Today 1998,3, 552.

Other linkers include members of a binding pair. In this arrangement,one binding pair member is attached to the flow channel interiorsurface. The other member of the binding pair is attached to the ligand(antiligand) one seeks to attach to the channel surface. Exemplarybinding pair members include biotin/avidin (or streptavidin) andantigen/antibody.

Depending upon the composition of the flow channel, it sometimes isnecessary to derivatize the flow channel inner surface so that thebinding pair member can be attached. As described supra in the sectionon channel coatings, a variety of agents can be coated onto the interiorchannel surface to introduce functionality. Examples include, but arenot limited to, silanizing reagents (e.g., 3-aminopropyltriethoxysilane,N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, dimethylchlorosilane orhexamethyldisilazane) which form self-assembled monolayers on theelastomer surface (which can be used to treat individual flow channels),and photochemical crosslinking reagents to create patterns of reactivegroups on the elastomer surface (e.g., aryl azide derivatives orquinone-based derivatives). Elastomer surfaces can also be subjected tooxygen plasma treatment or passively modified by creating multiplelayers of agents on the interior surface as described above in thesection on channel coatings.

Washing of complexes can also be achieved in a variety of ways. Oneapproach is to trap the complexes within a cage and then flow the washsolution (s) through the cage. Another option is to transfer complexesfrom a pen to a chamber containing wash solution. After being contactedwith the wash solution, the complexes are transported back to the penand a new wash solution introduced into the chamber. The complexes canthen subsequently be returned to the chamber for additional washing.This process can be repeated as many times as necessary.

3. Homogeneous Assays

The binding assays conducted with the microfluidic devices providedherein can also be conducted in homogeneous formats. In the homogeneousformats, ligands and antiligands are contacted with one another insolution and binding complexes detected without having to removeuncomplexed ligands and antiligands.

FP and FRET: Two approaches frequently utilized to conduct homogenousassays are fluorescence polarization (FP) and FRET assays, which aredescribed supra. FP assays can be conducted in a homogenous formatbecause the method is sensitive to tumbling rates of the fluorescentlylabeled entity. If a labeled ligand is free is solution its tumblingrate is significantly higher than that for a ligand that has becomecomplexed to an antiligand, especially if the antiligand is amacromolecule (see, e.g., Chen et al. (1999) Genome Research 9: 492-8;and U.S. Pat. No. 5,593,867 to Walker et al.).

FRET assays can sometimes be performed in a homogenous format because indirect binding assays the donor and acceptor fluorophores borne by theligands and antiligands initially are generally far enough apart thatminimal energy transfer occurs. However, once a ligand/antiligandcomplex is formed, the donor and acceptor labels are broughtsufficiently close to one another such that energy transfer, whichtransfer can be detected.

Confocal Microscopy As indicated supra, certain systems utilize amicrofluidic device as provided herein utilizing confocal microscopy asa detection method. Such arrangements can be used to conduct bindingassays in a homogenous format. Certain of these assays generally involvemeasuring amounts of bound and free signal utilizing confocal microscopyto distinguish between labeled ligand that is part of a complex andunbound labeled ligand. Confocal microscopy allows this distinction tobe made by taking advantage of the fact that with confocal microscopyone can confine detection of an illuminated species to a narrow objectplane. Thus, one can essentially view a thin slice of a sample, forexample. The same result can be achieved using conventional microscopy(i.e., non-confocal microscopy) by sequentially viewing different depthsin the sample, albeit in a less convenient manner.

Hence, in certain assays utilizing the devices described herein, testcompounds are screened for their ability to inhibit or promote bindingbetween a labeled ligand (e.g., a fluorescently labeled ligand) and atarget molecule that is present on the surface of a cell or support. Theeffect, if any, of the test compound on binding between the labeledligand and target molecule is monitored by determining the amount oflabeled ligand complexed with the cell or support in the presence of thetest compound. Unbound ligand appears as a background of relativelyconstant signal. Ligand that is part of a complex, in contrast, appearsas regions of increased fluorescence against such a background. Thus, inassays utilizing fluorescently labeled ligands, for example, the amountof fluorescence associated with individual cells or supports is totaledto obtain a value that is representative of the amount of bindingbetween the labeled ligand and target. This amount is compared to theamount of free fluorescence to obtain a value that is a measure of theinhibitory or enhancing effect of the test compound.

Assays of this type are preferably conducted using laser scanningconfocal microscopy. Such microscopes are commercially available fromBiometric Imaging Inc. (Mountain View, Calif.), for example. Otherconfocal microscopes that are suitable in certain applications aredescribed in U.S. Pat. Nos. 5,032,720; 5,120,953; 5,260,578; 5,304,810;5,283,684; and 5,162,946. The use of confocal microscopy in bindingassays is also discussed in U.S. Pat. No. 5,876,946.

Scintillation Proximity Assays: Other homogenous assays that can beperformed with the provided devices utilize supports (e.g., polymericbeads) that are coated or impregnated with scintillant and that bear aligand that can bind to a radiolabeled target molecule in a sample. Uponbinding of the ligand to the target, the radiolabel activates thescintillate such that it emits a detectable signal. The level of theemitted signal is a measure of the amount of ligand complexed with thetarget in the sample. Assays of this type are described further in U.S.Pat. No. 4,568,649, for example. Beads for conducting such assays areavailable from Amersham Corp. (Arlington Heights, Ill.).

Europium Cryptate Methods: The microfluidic devices can also be used inbinding assays that utilize ligands labeled with an energy-donatinglabel having a long-lived fluorescent state and targets that are labeledwith an energy-accepting label that has a short fluorescent excitedstate. Often such assays are performed using ligands labeled withEuropium-cryptate (the energy donating label) and a target that islabeled with an energy-accepting protein such as allophycocyanin (thelabel with the short fluorescent excited state). Energy transfer occursbetween these labels once they are brought within close proximity (e.g.,less than 7 nm apart) such as occurs upon formation of a complex thatcontains these two labels. The assay itself involves exciting theEu-cryptate with a pulsed laser. The fluorescent emission from thislabel continually re-excites the allophycocyanin. Fluorescence from thisprotein can be measured utilizing time resolved fluorescent techniques.

4. Assays for Compounds that Inhibit Binding Interactions

The microfluidic devices can also be utilized in a competitive formatsto identify agents that inhibit the interaction between known bindingpartners. Such methods generally involve preparing a reaction mixturecontaining the binding partners under conditions and for a timesufficient to allow the binding partners to interact and form a complex.In order to test a compound for inhibitory activity, the reactionmixture is prepared in the presence (test reaction mixture) and absence(control reaction mixture) of the test compound. Formation of complexesbetween binding partners is then detected, typically by detecting alabel borne by one or both of the binding partners. The formation ofmore complexes in the control reaction then in the test reaction mixtureat a level that constitutes a statistically significant differenceindicates that the test compound interferes with the interaction betweenthe binding partners. The order of addition of reactants can be variedto obtain different binding information concerning the compounds beingtested. For example, test compounds that interfere with the interactionbetween binding pair members can be identified by conducting thereaction in the presence of the test compound, i.e., by introducing thetest compound into the reaction mixture prior to or simultaneously withthe binding pair members. Alternatively, test compounds capable ofdisrupting preformed complexes can be identified by adding the testcompound to the reaction mixture after the complexes have been formed.This latter type analysis enables one to identify compounds that have ahigher binding constant then one of the members of the binding pair andthus is able to displace that binding pair member from the complex.

5. Immunological Assays

Immunological assays are one general category of assays that can beperformed with the microfluidic devices provided herein. Certain assaysare conducted to screen a population of antibodies for those that canspecifically bind to a particular antigen of interest. In such assays, atest antibody or population of antibodies is contacted with the antigen.Typically, the antigen is attached to a solid support. The support canbe a mobile support such as a bead or the interior wall of a flowchannel, for example. If mobile supports are utilized, the supports aretypically retained within a holding space during washing and/ordetection to facilitate the analysis. If the antigen is attached to aflow channel wall, typically the antigens are attached within thedetection region or a holding zone to aid detection. Other assays areconducted to examine a sample to determine if an analyte of interest ispresent by detecting binding between an antibody that specificallyrecognizes the analyte and the analyte. In assays such as this, often itis the antibody that is attached to a support (e.g., a mobile support orthe wall) and a solution containing potential antigens contacted withthe immobilized antibody. In both types of assays, however, either theantigen or antibody can be immobilized. The screens can also beconducted in a homogeneous format in which complexes are formed insolution and detected without further separation. This can beaccomplished, for example, by monitoring labeled antigen by fluorescencepolarization. Because the tumbling rate of labeled antigen will beconsiderably faster than the tumbling rate once the antigen is bound byantibody, labeled complexes can be distinguished from labeled antigen.

Immunological assays can be conducted in a variety of different formats.For example, the assays can involve direct binding between antigen andantibody, the so-called sandwich assay, enzyme linked immunosorbentassays (ELISA) and competitive assays. In an ELISA assay, for example, acapture antibody that specifically binds to the analyte of interest isattached to a solid support. As indicated supra, attachment can be to asupport or a surface of a flow channel that is located at a position inthe flow channel that can be monitored by the detector. A solutionpotentially containing the analyte of interest is then introduced into aflow channel and contacted with the immobilized capture antibody to forma binary complex. A second antibody (a detection antibody) thatrecognizes another portion of the analyte than the capture antibody isthen contacted with the binary complex to form a ternary complex. Thedetection antibody includes an assayable enzyme. Thus, formation of theternary complex can be detected by introducing the appropriate enzymesubstrate into the flow channel and allowed to contact any ternarycomplex. Signal produced in association with the enzyme catalyzedformation of product is detected by the detector.

As discussed supra, capture antibodies can be attached to flow channelinterior surfaces via functional groups borne by the antibody (e.g.,amino, carboxyl, sulfhydryl, hydroxyl) and complementary groups on thechannel surface or introduced by derivatization.

E. Enzyme Assays

1. General

Utilizing the microfluidic devices provided herein, a variety ofenzymatic assays can be performed. Such enzymatic assays generallyinvolve introducing an assay mixture containing the necessary componentsto conduct an assay into the various branch flow channels. The assaymixtures typically contain the substrate (s) for the enzyme, necessarycofactors (e.g., metal ions, NADH, NAPDH), and buffer, for example. If acoupled assay is to be performed, the assay solution will also generallycontain the enzyme, substrate (s) and cofactors necessary for theenzymatic couple.

Solutions containing the enzyme to be assayed are subsequently mixedwith the assay solution. Depending upon the configuration of themicrofluidic device, this can be accomplished in a number of differentways. For example, the assay solution can be retained in a holding spaceand then mixed with enzyme introduced into a branch flow channel.Alternatively, using a device such as that illustrated in FIG. 12 or 19,assay solution can be introduced into a branch flow channel via the mainflow channel and a portion retained in the holding space. Differentsolutions containing enzymes can subsequently be introduced into thedifferent branch flow channels to partially fill the chamber within thebranch flow line. The assay solution retained in the holding space canthen be transported to the chamber to initiate reactions and theresulting mixture returned to the holding section or to the detectionregion to monitor enzymatic activity. Assays can also be conducted byfirst introducing enzyme solutions into the different branch flowchannels and then introducing assay solutions into the various branchflow channels. However, it is usually easier to introduce the commonreactant, in this case the assay solution, into the main flow channelwhich allows for the facile diversion of assay solution into each of thebranch flow channels, than to separately introduce the common reactantinto each branch flow channel.

If a device such as shown in FIG. 12, 13 or 19 is utilized, common assayreagents can be introduced via the main flow channel (s) and differentsamples via the branch flow channels.

The mode of detection will vary depending upon the nature of the productgenerated. Often enzymatic reactions involve monitoring the appearanceof a detectable product or the disappearance of a detectable substratethat absorb or emit light at a particular wavelength. In such instances,the detector is able to detect absorption or emission at thatwavelength. Fluorescence polarization can also be utilized to conductenzyme assays. As noted supra, fluorescence polarization involvesdetecting differences in signals resulting from differential tumblingrates for large and small labeled agents. One strategy is to use afluorescently labeled substrate that bears a member of a binding pair.The assay solution also includes a macromolecule that bears the othermember of the binding pair. In the absence of enzyme, the fluorescentlylabeled substrate becomes attached to the macromolecule via interactionbetween the binding pair members. However, if enzyme is present, thesubstrate is cleaved, producing a small labeled substrate that producesa significantly different polarization signal then the fluorescent labelwhen attached to the macromolecule (see, e.g., Levine, et al. (1997)Anal. Biochem. 247: 83-8). A variety of screening assays can also beconducted with the microfluidic devices provided herein to identifycompounds that modulate (i.e., activate or inhibit) an enzymaticactivity of interest. In general such compounds are generally screenedby contacting the enzyme with a substrate in the presence and absence ofthe compound being screened under conditions conducive to the activityof the enzyme. The resulting reactant mixture is then assayed for thepresence of reaction product or a decrease in substrate concentration.In some instances, this amount is compared to a control reactionconducted in parallel with the test reaction. The control reaction caninvolve contacting the enzyme in the absence of test compound or in thepresence of a known inhibitor, for example. In operation, the foregoingmethods describing enzyme assays generally apply, except that the assaysolution also includes the compound under test. Alternatively, the testcompound can also be introduced separately.

Another option is to utilize devices that include the elastomeric mixersdescribed supra. These mixers can be useful for ensuring adequate mixingbetween assay solution enzyme solution before detection is initiated.Additionally, detection can be continued over time to follow the rate ofreaction with time. Such information can be utilized to determinekinetic values.

2. Methods wherein Generated Product is Detected by Optical Means

Heterogeneous Time-Resolved Fluorescence (HTRF). HTRF is one detectionscheme that can be used to conduct enzymatic assays. For example,tyrosine kinases can be assayed using HTRF. In such assays, a kinasepeptide substrate labeled with a fluorescent acceptor molecule (e.g.,XL665 available from Packard Biosciences) is incubated with the kinaseand inhibitors. Kinase activity is detected by adding a EuK-labeledanti-phosphotyrosine antibody (available from Packard Biosciences). Uponbinding of the antibody to the phosphotyrosine residue, the acceptor anddonor are brought into close contact. Excitation at 337 nm then resultsin an increase of fluorescence at 665 nm measured 50 μs afterexcitation, and an increase in the F₆₆₅/F₆₂₀ ratio.

Such assays can be conducted with the high throughput screening devicesdisclosed herein. For instance, using the device shown in FIG. 13,common reaction components can be introduced into the main flow channels(i.e., channels A, B and C) in any order, and then mixed withinhibitors. Thus, for example, substrate, inhibitor and antibody can beintroduced into the same or different main flow channels. Samples can beintroduced into the branch flow channels (i.e., channels 1, 2 and 3). Assamples are transported down the branch flow channels, they become mixedwith the substrate, inhibitor and antibody located in the chambers.After incubation for a suitable time, the reaction is measured using anHTRF approach with excitation at 337 nm and ratiometric measurement ofF₆₆₅/F₆₂₀. Detection is then performed in the chamber at which all assaycomponents have been added or further downstream in a separate detectionsection.

Fluorescence resonance energy transfer (FRET). Methods utilizing FRETare another common way to detect enzymatic activity; such methods can beperformed with the present microfluidic devices. Certain FRET-basedmethods utilize fluorescently labeled proteins as part of the detectionstrategy. For example, FRET can be generated when green fluorescentprotein (GFP) and blue fluorescent protein (BFP) are covalently linkedtogether by a short peptide. Cleavage of this linkage by a proteasecompletely eliminates the FRET effect. Such an approach can be used withcaspase-3 (CPP32), an important cellular protease activated duringprogrammed cell death, for example. An 18 amino acid peptide containinga CPP32 recognition sequence, DEVD, can be used to link GFP and BFPtogether. CPP32 activation can be monitored by FRET assay during theapoptosis process (see, e.g., Xu, X., A. L. Gerard, et al. (1998).Nucleic Acids Res 26:2034-5.

Fluorescence polarization. FP is another useful enzymatic detectionmethod that can be utilized with the devices and assay methods describedherein. As indicated supra, FP can be used to study a variety ofligand/antiligand interactions. For example, this approach can be usedto study receptor-ligand interactions (surface and nuclear receptors,cytokine & chemokines and their receptors) as well as enzymaticreactions (e.g. serine/threonine and tyrosine kinases and hydrolyticreactions such as proteases and hydrolases). As a specific example, theactivity of Protein Kinase C (PKC) family members (phosphorylation ofserine and threonine residues) can be assayed with the presentmicrofluidic devices. The activity of Protein Kinase C (PKC) familymembers is critical to the normal regulation of many biologicalmechanisms, including the modulation of membrane structure and skeletalreorganization, receptor desensitization, transcriptional control, cellgrowth and differentiation, and mediation of immune response. PKCs alsoplay a role in memory, learning and long-term potentiation.

An example of a specific PCK assay is a competition assay in which afluorescent phosphopeptide tracer and the nonfluorescent phosphopeptidesgenerated during a PKC reaction compete for binding to anantiphosphoserine antibody. In a reaction mixture containing nophosphopeptide product, the fluorescent tracer is bound by the antibodyand the emission signal is polarized. However, in a reaction mixturecontaining phosphopeptide product, the fluorescent tracer is displacedfrom the antibody and the emission signal becomes depolarized. Using amicrofluidic device such as depicted in FIGS. 12 and 13, components ofthe reaction, including the fluorescent and competitive substrates andinhibitors, can be introduced via the main flow channel (s) and mixedrapidly with potential inhibitors introduced into the branch flowchannels. Sequestration of reactions in rotary mixers enables aliquotsto be withdrawn, quenched as necessary and moved into a detection regionfor determination of polarization. Sampling of aliquots allows kineticmeasurements to be measured.

3. Capture of Enzyme Product

The devices can be arranged to include a material that selectively bindsto an enzymatic product that is produced. In some instances, thematerial has specific binding affinity for the reaction product itself.Somewhat more complicated systems can be developed for enzymes thatcatalyze transfer reactions. Certain assays of this type, for example,involve incubating an enzyme that catalyzes the transfer of a detectablemoiety from a donor substrate to an acceptor substrate that bears anaffinity label to produce a product bearing both the detectable moietyand the affinity label. This product can be captured by material thatincludes a complementary agent that specifically binds to the affinitylabel. This material typically is located in a detection region suchthat captured product can be readily detected. In certain assays, thematerial is coated to the interior channel walls of the detectionsection; alternatively, the material can be a support located in thedetection region that is coated with the agent.

One specific example of such an approach is one in which the affinitylabel is a metal chelating agent (e.g., a plurality of histidineresidues) and the capture material is a metal-derivatized material. Evenmore specifically, in certain assays, the detectable moiety that istransferred is radiolabeled such that the reaction product bears theradiolabel and the metal chelating agent. The capture material in thisparticular assay is also coated or impregnated with scintillant. Thus,once the reaction product becomes bound to the metal-derivatized capturematerial via the metal chelating agent, the radiolabel causes thescintillant to emit a signal that can be detected. Systems utilizingsuch an approach are discussed further, for example, in U.S. Pat. No.6,146,842. One advantage of this particular assay but also other assaysof this general type is that they often can be conducted in a homogenousformat, thus allowing detection to proceed without the need to removeother reactants. Using assays of this type, test compounds can also beincluded in the assay mixtures to determine their ability to enhance orinhibit the transfer activity.

F. Receptor Activation and Second Messenger Assays

1. Ligand-Receptor FCS Assays

Certain assays utilizing the present devices are conducted with vesiclesrather than cells. Once example of such an assay is a G-protein coupledreceptor assay utilizing fluorescent correlation spectroscopy (FCS).Membrane vesicles constructed from cells that over-express the receptorof interest are introduced into a main flow channel. Vesicles can eitherbe premixed with inhibitor and introduced via branch flow channels orvia one of the main flow channels prior to being mixed with afluorescent natural ligand which is also introduced by a main flowchannel. Components are allowed to incubate for the desired time andfluorescent signals analyzed directly in the flow chamber using an FCSreader such as the Evotec/Zeiss Confocor (a single or dual photoncounting device).

2. Ligand-Receptor FRET Assays

FRET assays can also be utilized to conduct a number of ligand-receptorinteractions using the devices disclosed herein. For example, a FRETpeptide reporter can be constructed by introducing a linker sequence(corresponding to an inducible domain of a protein such as aphosphorylation site) into a vector encoding for a fluorescent proteincomposed of blue- and red-shifted GFP variants. The vector can be abacterial (for biochemical studies) or a mammalian expression vector(for in vivo studies). For instance, a FRET peptide reporter composed ofa region in the cAMP-responsive element binding protein (CREB)designated KID (kinase-inducible domain) can be utilized (see, e.g.,Nagai, Y. et al. (2000) Nat. Biotechnol. 18:313-316). Thekinase-inducible domain contains a phosphorylation site at Ser 100 forprotein kinase A (PKA). Upon phosphorylation, there is afluorescence-activated energy transfer (FRET) between donor (Blue GFP)and acceptor (green GFP). cAMP induced, PKA-mediated phosphorylation ofKID on SER133 leads to a conformational change that decreases FRETbetween the donor and acceptor. The ratio between the emissions at 450nm and 510 nm is increased in response to KID phosphorylation. A controlpeptide containing the fluorescent protein without the linker sequencecan be utilized in control reactions.

For an in vitro assay, the FRET peptide reporter fusion protein ispurified from bacteria and a standard biochemical assay (i.e. kinaseassay) is performed using the device in FIG. 13 or 19, for example. Foran in vivo assay, a mammalian expression vector that encodes the FRETpeptide reporter is transfected into mammalian cells as described inNagai et al. (Nat. Biotechnol. 18: 313-316 (2000)).

3. Nuclear Receptors

FRET Assays: Assays of nuclear receptors can also be performed with thepresent microfluidic devices. For example, FRET-based assays forco-activator/nuclear receptor interaction can be performed. As aspecific example, such assays can be conducted to detect FRETinteractions between: (a) a ligand binding domain of a receptor taggedwith CFP (cyan fluorescent protein, a GFP derivative), and (b) areceptor binding protein (a coactivator) tagged with the Yellowfluorescent protein (YFP). Interaction between these components that areabolished by receptor antagonists can be detected by the loss of FRET.Further details of such methods are provided by Llopis et al. (2000)Proc. Natl. Acad. Sci. USA 97: 4363-8.

Such cell-based assays can be implemented in the present microfluidicdevices such as that shown in FIGS. 12 and 13. Cells transfected withconstructs in which the relevant coactivator and nuclear receptor ligandbinding domain are tagged with acceptor CFP and donor BFP. Cells areincubated with potential inhibitors and ratiometric measurements at eachof the emission maxima of the donor and acceptor are made.

Fluorescence polarization (FP): FP can be utilized to develop highthroughput screening (HTS) assays for nuclear receptor-liganddisplacement and kinase inhibition. Because FP is a solution-based,homogeneous technique, there is no requirement for immobilization orseparation of reaction components. In general, the methods involve usingcompetition between a fluorescently labeled ligand for the receptor andrelated test compounds. Examples of such assays are discussed by Parker,G. J., T. L. Law, et al. (2000) J Biomol Screen 5:77-88.

G. Cell Reporter Assays

A number of different cell reporter assays can be conducted with theprovided microfluidic devices. One common type of reporter assay thatcan be conducted include those designed to identify agents that can bindto a cellular receptor and trigger the activation of an intracellularsignal or signal cascade that activates transcription of a reporterconstruct. Such assays are useful for identifying compounds that canactivate expression of a gene of interest. Two-hybrid assays are anothermajor group of cell reporter assays that can be performed with thedevices. The two-hybrid assays are useful for investigating bindinginteractions between proteins. These are discussed in greater detail inthe following sections.

Often cell reporter assays are utilized to screen libraries ofcompounds. Although a variety of microfluidic devices including thevarious components disclosed herein can be used, a device such as thatshown in FIG. 12 can be utilized in the high throughput screening of alibrary of compounds for their ability to activate a receptor orinfluence binding between binding proteins. In general such methodsinvolve introducing the cells into the main flow channel so that cellsare retained in the chambers located at the intersection between themain flow channel and branch channels. Different test agents (e.g., froma library) can then be introduced into the different branch channelswhere they become mixed with the cells in the chambers.

Alternatively, cells can be introduced via the main flow channel andthen transferred into the branch channel, where the cells are stored inthe holding areas. Meanwhile, different test compounds are introducedinto the different branch flow channels, usually to at least partiallyfill the chambers located at the intersection of the main and branchflow channels. The cells retained in the holding area can be released byopening the appropriate valves and the cells transferred to the chambersfor interaction with the different test compounds. Once the cells andtest compounds have been mixed, the resulting solution is returned tothe holding space or transported to the detection section for detectionof reporter expression. The cells and test agents can optionally befurther mixed and incubated using mixers of the design set forth above.

1. Receptor Binding and Gene Activation

The cells utilized in screening compounds to identify those able totrigger gene expression typically express a receptor of interest andharbor a heterologous reporter construct. The receptor is one whichactivates transcription of a gene upon binding of a ligand to thereceptor. The reporter construct is usually a vector that includes atranscriptional control element and a reporter gene operably linkedthereto. The transcriptional control element is a genetic element thatis responsive to an intracellular signal (e.g., a transcription factor)generated upon binding of a ligand to the receptor under investigation.The reporter gene encodes a detectable transcriptional or translationalproduct. Often the reporter (e.g., an enzyme) can generate an opticalsignal that can be detected by a detector associated with a microfluidicdevice. General cell reporter systems are discussed in U.S. Pat. No.5,436,128. In addition to their use in identifying compounds that areligands of receptors that trigger gene activation, the methods can alsobe used to identify compounds that are agonists or antagonists ofreceptors of interest.

A wide variety of receptor types can be screened. The receptors oftenare cell-surface receptors, but intracellular receptors can also beinvestigated provided the test compounds being screened are able toenter into the cell. Examples of receptors that can be investigatedinclude, but are not limited to, ion channels (e.g., calcium, sodium,potassium channels), voltage-gated ion channels, ligand-gated ionchannels (e.g., acetyl choline receptors, and GABA (gamma-aminobutyricacid) receptors), growth factor receptors, muscarinic receptors,glutamate receptors, adrenergic receptors, dopamine receptors (see,e.g., U.S. Pat. Nos. 5,401,629 and 5,436,128). Additional receptorsinclude the G-protein coupled receptors, specific examples of whichinclude substance K receptor, the angiotensin receptor, the α- andβ-adrenergic receptors, the serotonin receptors, and PAF receptor (see,e.g., Gilman (1987) Ann. Rev. Biochem. 56:625-649).

A number of different reporters can be used to detect gene activation.Certain reporter gene encode proteins that are inherently detectable.One specific example of such a reporter is green fluorescent protein.Fluorescence generated from this protein can be detected with variouscommercially-available fluorescent detection systems. Other reporterscan be detected by staining. Most commonly, however, the reporter is anenzyme that generates a detectable signal when contacted with anappropriate substrate. Often the reporter is an enzyme that catalyzesthe formation of a detectable product. Suitable enzymes include, but arenot limited to, proteases, nucleases, lipases, phosphatases andhydrolases. Typically, the reporter encodes an enzyme whose substratesare substantially impermeable to eukaryotic plasma membranes, thusmaking it possible to tightly control signal formation. Specificexamples of suitable reporter genes that encode enzymes include, but arenot limited to, CAT (chloramphenicol acetyl transferase; Alton andVapnek (1979) Nature 282: 864-869); luciferase (lux); β-galactosidase;LacZ; β-glucuronidase; and alkaline phosphatase (Toh, et al. (1980) Eur.J. Biochem. 182: 231-238; and Hall et al. (1983) J. Mol. Appl. Gen. 2:101), each of which are incorporated by reference herein in itsentirety. Other suitable reporters include those that encode for aparticular epitope that can be detected with a labeled antibody thatspecifically recognizes the epitope.

The cells utilized in the cell based assay can include any of thosedescribed supra. In general, the cells must express the receptor ofinterest. The receptor can be an endogenous receptor or a receptorexpressed from a heterologous construct that includes the gene thatencodes for the receptor of interest. In the latter instance, the cellmust be able to transfected with the construct and able to express theheterologous receptor.

2. Two Hybrid Assays

Another general category of cell assays that can be performed is the twohybrid assays. In general, the two-hybrid assays exploit the fact thatmany eukaryotic transcription factors include a distinct DNA-bindingdomain and a distinct transcriptional activation domain to detectinteractions between two different hybrid or fusion proteins. Thus, thecells utilized in two-hybrid assays include the construct (s) thatencode for the two fusion proteins. These two domains are fused toseparate binding proteins potentially capable of interacting with oneanother under certain conditions. The cells utilized in conductingtwo-hybrid assays contain a reporter gene whose expression depends uponeither an interaction, or lack of interaction, between the two fusionproteins.

In standard two-hybrid systems, interaction between the two bindingproteins results in expression of the reporter gene (see, e.g., Fields,S, and Song, O. (1989) Nature 340: 245; Bartel, P. L., and Fields, S.(1995) Methods in Enzymology 254: 241-263; Heery, D. M., et al. (1997)Nature 387: 733-736; and Bartel, et al. (1993) Biotechniques 14: 920).Reverse hybrid systems, in contrast, are designed such that interactionbetween the two binding proteins suppresses reporter expression. Inthese systems, reporter expression is triggered when a compound inhibitsthe interaction between the two binding proteins (see, e.g., PCTpublication WO 95/26400). Two-hybrid assays can be utilized to establishinteractions between two known proteins or to search a genomic or cDNAlibrary for proteins that interact with a target protein.

H. Monitoring Cell Membrane Potential

A variety of methods to assay for cell membrane potential can beconducted with the microfluidic devices disclosed herein. In general,methods for monitoring membrane potential and ion channel activity canbe measured using two alternate methods. One general approach is to usefluorescent ion shelters to measure bulk changes in ion concentrationsinside cells (see below). The second general approach is to use of FRETdyes sensitive to membrane potential (see. e.g., U.S. Pat. Nos.6,124,128 and 5,981,200). Ion channel measurements are typicallyconducted with whole cells that encode endogenous ion channel componentsor expressing heterologous constructs that encode ion-channelcomponents. A discussion of such assay is provided by Takahashi, et al.(1999) Physiol. Rev. 79: 1089-125 and Gonzalez, et al. (1997) Chem.Biol. 4: 269-77. As a specific example of such methods that can beutilized with the present devices, certain fluorescence-based assaysinvolve the detection of cell membrane potentials based on the transferof fluorescence resonance energy between fluorescently labeledphospholipids. The measurement of changes in membrane potential dependsupon the disruption of FRET from coumarin-labeled donor lipids to oxonolacceptors that electrophorese from one face of the membrane to the otherin response to membrane potential. For instance, certain assays utilizea coumarin-labeled phosphatidylethanolamine donor and abis(1,3-dihexyl-2-thiobarbiturate) trimethineoxonol acceptor. Thisparticular combination is highly sensitive (fluorescence ratiochange >50% per 100 mV). Response can also be speeded several-fold bylengthening the mobile dye to the pentamethineoxonol analog.

In operation with the present devices, cells are preloaded with FRETdyes and introduced through a common reagent port. Potential inhibitorsare loaded through branch flow channels. Cells can be trapped in a penand flushed with the inhibitors. The ion channel is then stimulated toopen by the application of an appropriate stimulus (ligand or ion). FRETmeasurements are made at appropriate wavelengths.

I. Cell Proliferation Assays

The microfluidic devices disclosed herein can be utilized to conduct avariety of different assays to monitor cell proliferation. Such assayscan be utilized in a variety of different studies. For example, asdescribed further infra, the cell proliferation assays can be utilizedin toxicological analyses, for example. Cell proliferation assays alsohave value in screening compounds for the treatment of various cellproliferation disorders including tumors. Compounds identified as havingactivity as inhibitors of cell proliferation can be utilized to inhibitmitogenesis, inhibit angiogenesis and to activate the complementpathway, including activation of killer cells.

The ability to conduct assays of angiogenesis is of value becauseangiogenesis refers to the formation of blood and lymph vessels.Angiogenesis plays an important role in a number of differentphysiological processes including embryonic development, wound healingand the development of the endometrium after menstruation. Abnormalangiogenesis is correlated with a number of diseases including, forexample, diabetic retinopathy, rheumatoid arthritis, hemangiomas and thegrowth of solid tumors. It can also be involved in coronary arterydisease and restenosis following angioplasty. Thus, compounds identifiedas inhibitors can serve as candidates in the treatment of thesediseases, as well as various dermatological disorders such as psoriasisthat have an angiogenic component. One approach for rapidly screeningfor compounds that modulate cell proliferation is to determine acidphosphatase levels, as the activity of this enzyme is indicative of cellproliferation and/or cell survival. Any of a number of establishedphosphatase assays can be utilized to conduct cell proliferation assays.One example of such assays are fluorogenic enzyme assays that utilizefluorescently labeled substrates that generate a fluorescent signal uponcleavage by a phosphatase. One category of suitable substrates arevarious benzothiazole substrates which when hydrolyzed by acidphosphatase generate a fluorescent product that can be detected. Suchsubstrates are described, for example, in U.S. Pat. Nos. 5,424,440 and5,972,639.

Hence, certain assay methods of this type involve retaining cells withina cell cage as described supra. A lysing agent is then introduced intothe microfluidic device and pumped to the cell cage to lyse the capturedcells. The substrate for the phosphatase enzyme such as thefluorescently labeled benzothiazole compounds described above can beintroduced together with the lysing agent or separately after the cellshave been lysed. Fluorescence can be detected within the cell pen or atanother detection section located elsewhere.

J. Toxicology/Cell Death Assays

The microfluidic devices disclosed herein can be utilized to perform avariety of different assays designed to identify toxic conditions,screen agents for potential toxicity, investigate cellular responses totoxic insults and assay for cell death. A variety of differentparameters can be monitored to assess toxicity. Examples of suchparameters include, but are not limited to, cell proliferation,monitoring activation of cellular pathways for toxicological responsesby gene or protein expression analysis, DNA fragmentation; changes inthe composition of cellular membranes, membrane permeability, activationof components of death-receptors or downstream signaling pathways (e.g.,caspases), generic stress responses, NF-kappaB activation and responsesto mitogens. Related assays are used to assay for apoptosis (aprogrammed process of cell death) and necrosis. Specific examples ofsuch assays follow.

1. Cell Proliferation; and Morphological and Permeability Changes

Cell Proliferation Certain toxicology assays involve monitoring cellproliferation. A variety of different assays can be conducted to assesscell proliferation. One specific method involves irreversibly labelingcell-surface and intracellular proteins with a fluorescent dye specificfor such proteins. Daughter cells generated during cell proliferationhave only half the amount of irreversibly bound dye as the parent. Thus,cell proliferation is characterized by a diminution of cellularfluorescence intensity. Examples of suitable fluorescent dyes to use insuch assays include, but are not limited to,carboxyfluorescein-diacetate succinimidyl ester, SNARF-1 and Marina Blue(available from Molecular Probes, Inc.).

Morphological Changes Apoptosis in many cell types is correlated withaltered morphological appearances. Examples of such alterations include,but are not limited to, plasma membrane blebbing, cell shape change,loss of substrate adhesion properties. Such changes are readilydetectable with a light microscope. Cells undergoing apoptosis can alsobe detected by fragmentation and disintegration of chromosomes. Thesechanges can be detected using light microscopy and/or DNA or chromatinspecific dyes.

Altered Membrane Permeability: Often the membranes of cells undergoingapoptosis become increasingly permeable. This change in membraneproperties can be readily detected using vital dyes (e.g., propidiumiodide and trypan blue). Similarly, dyes can be used to detect thepresence of necrotic cells. For example, certain methods utilize agreen-fluorescent LIVE/DEAD Cytotoxicity Kit #2, available fromMolecular Probes. The dye specifically reacts with cellular aminegroups. In necrotic cells, the entire free amine content is available toreact with the dye, thus resulting in intense fluorescent staining. Incontrast, only the cell-surface amines of viable cells are available toreact with the dye. Hence, the fluorescence intensity for viable cellsis reduced significantly relative to necrotic cells (see, e.g.,Haugland, 1996 Handbook of Fluorescent Probes and Research Chemicals,6th ed., Molecular Probes, OR and http://www.probes.com).

In the foregoing assays that require observation by light microscopy,the inverted optical microscope described supra can be utilized tomonitor cell morphology, for example. If desired, the cells can becontained within a holding space during detection. Assays requiringaddition of dyes can be conducted in a variety of ways. For example, thecells to be assayed can be introduced into branch flow channels and thenretained in a holding space. By holding the cells in a cage throughwhich fluids can flow, the necessary dyes can be flowed through thespace in which the cells are trapped. Test and control cells can beexamined in separate branch flow channels. Of course, cells can be mixedwith the necessary dyes in the chamber and mixer units and thentransferred to a detection section or holding space for observation.

2. Dysfunction of Mitochondrial Membrane Potential

Mitochondria are the main energy source in cells of higher organisms.These organelles provide direct and indirect biochemical regulation ofdiverse cellular processes. These process include the electron transportchain activity, which drives oxidative phosphorylation to producemetabolic energy in the form of adenosine triphosphate (i.e., ATP).Altered or defective mitochondrial activity can result in mitochondrialcollapse called the “permeability transition” or mitochondrialpermeability transition. Proper mitochondrial functioning requiresmaintenance of the membrane potential established across the membrane.Dissipation of the membrane potential prevents ATP synthesis and thushalts or restricts the production of a vital biochemical energy source.Consequently, a variety of assays designed to assess toxicity and celldeath involve monitoring the effect of a test agent on mitochondrialmembrane potentials or on the mitochondrial permeability transition. Oneapproach is to utilize fluorescent indicators (see, e.g., Haugland, 1996Handbook of Fluorescent Probes and Research Chemicals, 6th ed.,Molecular Probes, OR, pp. 266-274 and 589-594). Various non-fluorescentprobes can also be utilized (see, e.g., Kamo et al. (1979) J. MembraneBiol. 49: 105). Mitochondrial membrane potentials can also be determinedindirectly from mitochondrial membrane permeability (see, e.g., Quinn(1976) The Molecular Biology of Cell Membranes, University Park Press,Baltimore, Md., pp. 200-217). Further guidance on methods for conductingsuch assays is provided in PCT publication WO 00/19200 to Dykens et al.

In those instances in which fluorescent probes are utilized, thenecessary dyes can be contacted with the cells or mitochondrial membranepreparations in a number of different ways utilizing the presentmicrofluidic devices. For example, the dyes can be flowed through a cagein which cells or organelles are retained. Alternatively, cells and dyescan be mixed in one of the chambers or mixers, with detection occurringsubsequently within a detection region or within a cage.

3. Caspase Activation

Apoptosis is the term used to refer to the process of programmed celldeath and involves the activation of a genetic program when cells are nolonger needed or have become seriously damaged. This process occurs inthe cells of most higher eukaryotes and is necessary for normaldevelopment and the maintenance of homeostasis. It also serves as adefense mechanism, as it provides a means by which the body is able torid itself of unwanted and potentially dangerous cells including, forexample, cells infected with viruses, tumor cells and self-reactivelymphocytes.

Apoptosis involves a cascade of biochemical events and is under theregulation of a number of different genes. One group of genes act aseffectors of apoptosis and are referred to as theinterleukin-1.beta.converting enzyme (ICE) family of genes. These genesencode a family of cysteine proteases whose activity is increased inapoptosis. Thus, inhibitors of these enzymes can inhibit apoptosis. TheICE family of proteases is generically referred to as caspase enzymes.The “c” in the name reflects the fact that the enzymes are cysteineproteases, while “aspase” refers to the ability of these enzymes tocleave after aspartic acid residues.

Consequently, some assays for apoptosis are based upon the observationthat caspases are induced during apoptosis. Induction of these enzymescan be detected by monitoring the cleavage of specifically-recognizedsubstrates for these enzymes. A number of naturally occurring andsynthetic protein substrates are known (see, e.g., Ellerby et al. (1997)J. Neurosci. 17: 6165; Kluck, et al. (1997) Science 275: 1132; Nicholsonet al. (1995) Nature 376: 37; and Rosen and Casciola-Rosen (1997) J.Cell Biochem. 64: 50). Methods for preparing a number of differentsubstrates that can be utilized in these assays are described in U.S.Pat. No. 5,976,822. This patent also describes assays that can beconducted using whole cells that are amendable to certain of themicrofluidic devices described herein.

Certain caspase assays utilize FRET methodologies. In particular, asubstrate is prepared in which a donor fluorophore and an acceptorfluorophore are separated by an amino acid sequence that includes acleavage recognition site for the caspase protein of interest. Sincedifferent caspase cleave at different sequences, specific substrates canbe prepared for the particular caspase (s) of interest. Prior tocleavage, the donor and acceptor fluorophore are sufficiently close thatenergy transfer occurs. However, upon cleavage, the two labels are nolonger in an energy transfer relationship. The change in donor and/oracceptor fluorescence emissions can be detected as described supra.Further guidance on such an approach is discussed by Mahajan, et al.(1999) Chem. Biol. 6: 401-9; and Xu, et al. (1998) Nucl. Acids. Res. 26:2034-5. A specific example of a caspase assay is also provided infra inExample I.

4. Cytochrome c Release

In healthy cells, the inner mitochondrial membrane is impermeable tomacromolecules. Thus, one indicator of cell apoptosis is the release orleakage of cytochrome c from the mitochondria. Detection of cytochrome ccan be performed using spectroscopic methods because of the inherentabsorption properties of the protein. Thus, one detection option withthe present devices is to place the cells within a holding space andmonitor absorbance at a characteristic absorption wavelength forcytochrome c. Alternatively, the protein can be detected using standardimmunological methods (e.g., ELISA assays) with an antibody thatspecifically binds to cytochrome c (see, e.g., Liu et al. (1996) Cell86: 147). Detection in such instances can be accomplished as set forthabove in the section on immunological methods in the binding assaysection.

5. Assays for Cell Lysis

The microfluidic devices disclosed herein can also be utilized toperform assays to detect cell lysis. Such assays are related toapoptosis assays in that the final stage of cell death is typicallylysis of the cell. These assays can be utilized in a variety of studiesto examine the effects of agents or particular conditions on cells or ininvestigating the effect of particular compounds on cell health.

When cells die they typically release a mixture of chemicals, includingnucleotides, and a variety of other substances (e.g., proteins andcarbohydrates) into their surroundings. Some of the substances releasedinclude ADP and ATP, as well as the enzyme adenylate cyclase whichcatalyzes the conversion of ADP to ATP in the presence of excess ADP.Thus, certain assays involve providing sufficient ADP in the assaymedium to drive the equilibrium towards the generation of ATP which cansubsequently be detected via a number of different means. One suchapproach is to utilize a luciferin/luciferase system that is well knownto those of ordinary skill in the art in which the enzyme luciferaseutilizes ATP and the substrate luciferin to generate a photometricallydetectable signal.

Thus, the devices disclosed herein can be utilized to introduce testcompounds to cells to determine whether they cause the cells to lyse andthus have potential therapeutic value. For example, using tumor celllines, one can contact the cells with agents to screen for anti-tumoragents. Alternatively, test compounds can be introduced into the devicesand contacted with normal cells to assess the toxicity of suchcompounds. Thus, assays conducted with the devices can also be utilizedto conduct toxicological evaluations. Further details regarding certaincell lysis assays that can be performed with some of the microfluidicdevices described herein are set forth in PCT publication WO 00/70082.

K. Antimicrobial Assays

By contacting various microbial cells with different test compounds, onecan also utilize the devices provided herein to conduct antimicrobialassays, thereby identifying potential antibacterial compounds. The term“microbe” as used herein refers to any microscopic and/or unicellularfungus, any bacteria or any protozoan. Some antimicrobial assays involveretaining a cell in a cell cage and contacting it with at least onepotential antimicrobial compound. The effect of the compound can bedetected as any detectable change in the health and/or metabolism of thecell. Examples of such changes, include but are not limited to,alteration in growth, cell proliferation, cell differentiation, geneexpression, cell division and the like.

One approach for detecting an effect that a compound has on a cellinvolves utilizing a cell in which the natural promoter is replaced witha heterologous, regulatable promoter. This promoter also typically isoperably linked to a reporter gene (see supra). Replacement of thenatural promoter can be accomplished by homologous recombination orinsertional mutagenesis, for example. Thus, the level of metabolismafter a cell has been contacted with a test compound can be assessed byadding an inducer that activates the inserted promoter to triggerexpression of the linked reporter gene. Further discussion ofantimicrobial assays that can be conducted with whole cells which areamenable to the devices described herein is provided by PCT publicationWO 99/14311 and WO 01/07061.

L. Cell-Based Model Systems

The devices described herein are not limited to use in applications inwhich cells flow through channels. For some applications, certain flowchannels are segments thereof are coated with a matrix that promotescell adhesion and growth on the walls and floor of the channel. Suitablecoatings include, but are not limited to, poly-lysine, polyornithine,fibronectin, laminin and collagen. Coating with certain types of cellsallows different types of assays to be performed. Thus, for example, thehuman vascular system contains capillaries that have approximately 200μm internal diameter. A model of this capillary system can be created bypromotion of the coating of channels with human vascular epithelialcells (HUVECs). Using elastomeric pump devices such as described suprato create fluid flow through such coated channels (which is impossiblewith electrically-driven microfluidic devices), this in vitro modelprovides a system that is analogous to the in vivo system. Such a systemenables one to assay for a number of important activities, such as celladhesion in response to chemotactic activities, receptor-ligand bindingassays, cell activation and the like.

M. SNP Analysis

1. General

Relatively minor changes in the genome of an organism, including changesas small as a single nucleotide, can result in substantially differentphenotypes. For example, these changes or mutations can be responsiblefor a variety of different diseases, influence the efficacy of differenttherapeutic treatments and alter the pathogenicity of a microorganism orchange the resistance of a microorganism to therapeutics directedtowards it. Often such effects are the result of alteration of a singlenucleotide. Such alterations are generally referred to as singlenucleotide polymorphisms, or simply SNPs. The site at which an SNPoccurs is referred to as a polymorphic site or an allelic site. A numberof SNPs have been correlated with various human diseases (see, e.g.,Publication WO 93/02216 which provides an extensive list of such SNPs).Because SNPs appear regularly throughout the genome, they also serve asuseful genetic markers.

The ability to detect specific nucleotide alterations or mutations inDNA sequences has a number of medical and non-medical utilities. Forexample, methods capable of identifying nucleotide alterations provide ameans for screening and diagnosing many common diseases that areassociated with SNPs. Such methods are also valuable in identifyingindividuals susceptible to disease, those who could benefit fromprophylactic measures, and thus obtaining information useful in patientcounseling and education. Methods for detecting alterations andmutations have further value in the detection of microorganisms, andmaking correlations between the DNA in a particular sample andindividuals having related DNA. This latter capability can be useful inresolving paternity disputes and in forensic analysis.

2. Methods

Certain of the microfluidic devices provided herein can be utilized toconduct mini-sequencing reactions or primer extension reactions toidentify the nucleotide present at a polymorphic site in a targetnucleic acid. In general, in these methods a primer complementary to asegment of a target nucleic acid is extended if the reaction isconducted in the presence of a nucleotide that is complementary to thenucleotide at the polymorphic site. Often such methods are single basepair extension (SBPE) reactions. Such method typically involvehybridizing a primer to a complementary target nucleic acid such thatthe 3′ end of the primer is immediately adjacent the polymorphic site,or is a few bases upstream of the polymorphic site. The extensionreaction is conducted in the presence of one or more labelednon-extendible nucleotides (e.g., dideoxynucleotides) and a polymerase.Incorporation of a non-extendible nucleotide onto the 3′ end of theprimer prevents further extension of the primer by the polymerase oncethe non-extendible nucleotide is incorporated onto the 3′ end of theprimer.

More specifically, if one of the added non-extendible nucleotides iscomplementary to the nucleotide at the polymorphic site, then a labelednucleotide is incorporated onto the 3′ end of the primer to generate alabeled extension product. If, however, the non-extendible nucleotide isnot complementary to the nucleotide at the polymorphic site, thenlabeled nucleotide is not incorporated and there is no primer extension.Because the incorporated nucleotide is complementary to the nucleotideat the polymorphic site, extended primers provide an indication of whichnucleotide is present at the polymorphic site of target nucleic acids.

As noted supra, primers are chosen to have a sequence that willhybridize to the target nucleic acid such that the 3′ end of the primeris adjacent to the polymorphic site of the target. Preferably, the 3′end of the primer is immediately adjacent (but does not span) thepolymorphic site (i.e., the 3′ end hybridizes to the nucleotide justupstream of the polymorphic site). In some instances, methods can beperformed with primers that simply hybridize adjacent to the polymorphicsite but the 3′ end is several nucleotides upstream of the polymorphicsite. This is possible so long as none of the nucleotides on the targetnucleic acid located between the 3′ end of the primer and thepolymorphic site are the same as the nucleotide at the variant site. Theextension reaction mixture in such instances must also includenucleotides complementary to those nucleotides positioned between the 3′primer end and the polymorphic site.

Additional details and optional methods for conducing genotypingexperiments using primer extension reactions are discussed, for example,in U.S. Pat. Nos. 5,981,176; 5,846,710; 6,004,744; 5,888,819; 5,856,092;5,710,028; and 6,013,431; and in PCT publication WO 92/16657, each ofwhich is incorporated by reference in its entirety. With themicrofluidic devices disclosed herein, genotyping analyses can beconducted in a variety of different formats. For example, using a highthroughput screening device such as shown in FIG. 13, the assaycomponents needed to conduct an assay (e.g., buffer, primer, polymerase,and labeled dideoxynucleotide (s) ddNTPs) are introduced into the mainflow channel (s) (e.g., channels A, B and/or C in FIG. 13) and into thechambers located along the main flow channel. Different samplescontaining target nucleic acid (e.g., samples from differentindividuals) can be introduced into the branch flow channels (e.g.,channels 1, 2 and 3 in FIG. 13) where they become mixed with the assaycomponents. Each of the resulting mixtures can then be transportedthrough their respective branch flow channel and optionally into a mixeror holding area. The mixer or holding area can be positioned adjacent atemperature regulator to optimize the temperature for the extensionreaction (if any). Following incubation, incorporation of fluorescentlabel into the an extended primer can be detected in the detectionsection; the detection section can include the mixer or holding area orbe at some other location along the branch flow channel.

Other methods generally track the method just described, but in thisinstance, the same sample is investigated in a plurality of branch flowchannels. Thus, samples can be introduced into one of the main flowchannels (e.g., channel A in FIG. 13). However, in this instance, adifferent labeled non-extendible nucleotide is introduced into thedifferent branch flow channels that include sample from the sameindividual. Thus, for example, if a SNP is biallelic, one labelednon-extendible nucleotide complementary to one of the nucleotidespotentially at the polymorphic site is introduced into one branch flowchannel (e.g., channel 1 in FIG. 13); a non-extendible nucleotidecomplementary to the other potential nucleotide at the polymorphic siteis introduced into a second branch flow channel (e.g., channel 2 in FIG.13) that contains sample from the same individual. Of course, if a SNPis tetra-allelic or if the nature of the SNP is uncertain,non-extendible nucleotides for all four bases (e.g., ddATP, ddTTP,ddCTP, ddGTP) can be run in separate branch flow channels. Using amicrofluidic device of the type illustrated in FIG. 13, different assaycomponents can be individually introduced through the plurality of mainflow channels.

In some instances, the microfluidic device will include a separationmodule such as described supra to separate extension products fromextended primers and other reactants. In one configuration, each branchflow channel includes a section that includes a separation matrix ableto separate nucleic acids according to size. If the separation sectionincludes electrodes as described above, then separation can be by gelelectrophoresis. Another option is to transfer the reaction mixture toanother analytical device such as an HPLC or a nucleic acid analyzersuch as the MegaBACE analyzer from Molecular Dynamics.

N. Amplification Reactions

Related to the methods just described, the present devices can also beutilized to amplify and subsequently identify target nucleic acids inmultiple samples using amplification techniques that are wellestablished in the art. In general such methods involve contacting asample potentially containing a target nucleic acid with forward andreverse primers that specifically hybridize to the target nucleic acid.The reaction includes all four dNTPs and polymerase to extend the primersequences.

The devices disclosed herein such as those shown in FIGS. 12 and 13 canbe used to conduct amplification reactions as follows. Common reactantssuch as primers, dNTPs and polymerase are introduced into the main flowchannel (s) (e.g., channel A in FIG. 13). Different samples areintroduced into the branch flow channels (e.g., channels 1, 2 and 3 inFIG. 13). As described in the SNP screening section, the devices caninclude a variety of optional modules to further facilitate analysis.For example, branch flow channels can include mixers to adequately mixthe amplification mixtures and a temperature controller to regulatetemperature. This is particularly important with the amplificationreactions which undergo thermal cycling to allow for disassociation andreanneling of primers to target sequences.

Devices used in such amplification reactions can also include aseparation module. Often such separation modules are designed toseparate amplicons according to size. Thus, certain modules can includeelectrodes to allow for electrophoretic separations.

The present devices can be utilized in a wide variety of amplificationreactions. Examples of amplification reactions that can be conductedwith the device disclosed herein include, but are not limited to, (1)polymerase chain reaction (PCR) (see generally, PCR Technology:Principles and Applications for DNA Amplification (H. A. Erlich, Ed.)Freeman Press, NY, N.Y. (1992); PCR Protocols: A Guide to Methods andApplications (Innis, et al., Eds.) Academic Press, San Diego, Calif.(1990); Mattila et al. (1991) Nucleic Acids Res. 19: 4967; Eckert etal., PCR Methods and Applications 1: 17 (1991); PCR (McPherson et al.Ed.), IRL Press, Oxford; and U.S. Pat. Nos. 4,683,202 and 4,683,195,each of these being incorporated by reference in its entirety); (2)ligase chain reaction (LCR) (see, e.g., Wu and Wallace (1989) Genomics4: 560 and Landegren et al. (1988) Science 241: 1077); (3) transcriptionamplification (see, e.g., Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA86: 1173); (4) self-sustained sequence replication (see, e.g., Guatelliet al. (1990) Proc. Natl. Acad. Sci. USA, 87: 1874 (1990); and (5)nucleic acid based sequence amplification (NABSA) (see, e.g., Sooknanan,R. and Malek, L., (1995) Bio Technology 13: 563-65), each of which areincorporated by reference in their entirety. Further guidance regardingnucleic sample preparation is described in Sambrook, et al., MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, (1989), which is incorporated herein by reference in itsentirety.

The following examples are provided to further illustrate certainaspects of the invention, but are not to be construed so as to limit thescope of the invention.

EXAMPLE 1 Caspase Activity I. Background

Caspase-3 (also known as CPP32, apopain and Yama), a member of theinterleukin-1β converting enzyme, is one of the cystine proteases mostfrequently activated during the process of programmed cell death(apoptosis). Caspase-3 is essential for normal brain development, andhas been suggested to contribute to the molecular pathogenesis ofseveral neurological diseases. Thus, development of caspase inhibitorscan be used as potential therapeutic approaches for chronicneurodegenerative disorders. However, the assay model is not limited tothis protein.

II. Experimental Design

Using the PC12 pheochromocytoma cell line, a well-characterized model ofneuronal apoptotic cell death (see, e.g., Rukenstein et al. (1991) J.Neurosci. 11: 2552-2563; and Batistatou and Greene (1991) J. Cell Biol.115: 461-71), inhibitors of caspase-3 activity can be screened. Manyother cell lines known in the art can be used as well.

Screens are conducted, for example, using the device illustrated in FIG.13 according to the following steps. In should be recognized, however, awide variety of other devices can be utilized as well.

-   -   1. PC 12 cells are introduced through main flow channel A and        into the chambers. Potential inhibitors are introduced into the        branch flow channels 1, 2 and 3 and incubated with the cells in        the chambers for 10 minutes at 5% CO₂ and 37° C. to allow for        inhibitor/enzyme interaction.    -   2. Fluorogenic substrate (PhiPhiLux™, available from CalBiochem)        and stimulant (C2-Ceramide, available from Biomol Research        Laboratories, Inc.) are added to channels B and C, respectively.        PhiPhiLux™ is a peptide substrate for caspase-3 that has been        conjugated to two fluorophores. The substrate contains the        sequence GDEVDGI (caspase cleavage site is underlined). The        cleaved PhiPhiLux™ substrate has a green fluorescence with the        following fluorescence peak characteristics: λ_(ex)=505 nm and        λ_(em)=530 nm. When the folded peptide is cleaved, the        fluorophores provide a high intensity fluorescent signal at a        visible wavelength.    -   3. After 10 minutes, the cells are pumped into the chambers        positioned along main flow channel B and mixed with the cell        permeable Caspase-3 fluorogenic substrate PhiPhiLux™ at a        concentration of 10 μM and incubated for 20 minutes.    -   4. After 20 minutes, the cells are pumped further down their        respective flow channels to main flow channel C and mixed with        an apoptotic agent such as C2-Ceramide, a cell permeable        ceramide analog.    -   5. Caspase activity is assessed at several time points (e.g., 30        sec, 1 min, 5 min, etc.). After time t, fluorescent signals are        detected using an air-cooled argon laser with excitation at 488        nm and detection at 515 nm.

Assays such as this can be used to screen libraries of compounds forinhibitors or agonists of various enzymes. One can also use such a modelfor multiparametric analysis of signaling elements downstream of thetarget.

EXAMPLE 2 Screening NGF Agonists in PC12 Cells I. Background

Screens to identify agonists of receptors of interest can be performedusing the present devices. This example describes a method to screen foragonists of nerve growth factor receptor in PC12 cells (describedsupra). Docking of TrkA with the NGF receptor initiates receptordimerization, catalytic phosphorylation of cytoplasmic residues on thereceptor, and a cascade of signaling events (see, e.g., Kaplan andStephens (1994) J. Neurobio. 25: 1404-1417). Upon TrkA activation, thePI-3K/Akt pathway is activated which results in phosphorylation of Akt(see, e.g., Andjelkovic M. et al (1998) Eur. J. Biochem. 25: 195-200).Simultaneously, the MAP kinase pathway is activated which results inphosphorylation of Erk (Kaplan and Stephens, 1994).

II. Experimental Design

Although screens can be conducted with a variety of different devices,the following method is described with reference to the microfluidicdevice depicted in FIG. 13.

-   -   1. Cells expressing FRET reporters for TrkA, Erk and Akt are        added via main flow channel A. Thus, the cells express a TrkA        receptor that upon activation will generate a FRET a signal        different from that of the Erk and Akt FRET reporters.    -   2. Potential agonists are added via branch channels 1,2,3, etc.    -   3. After addition of the potential agonist: a. Receptor        activation is assessed at various time intervals (30 sec, 1 min,        5 min, etc.). A hit is a compound that acts like NGF and        stimulates the NGF receptor and the downstream signaling        elements (kinetics of the reaction should be the similar). b. If        the receptor is activated (phosphorylated), the kinetics of        receptor activation will be monitored (tyrosine phosphorylation        of the Trk receptor) via fluorescence. The cells express        proteins that FRET upon phosphorylation. Each protein (TrkA, Erk        and Akt) produces a FRET a specific signal different from one        another. c. Simultaneously, activation of the downstream        signaling elements are assessed and monitored. Activation of the        downstream elements (Akt, Erk) can be monitored via fluorogenic        substrates or FRET peptide reporters. Thus, for each hit, a        profile of activated elements (Trk receptor, Erk, Akt) can be        obtained, the profile including the duration and amplitude of        the signal.    -   4. After the cell is restored to its “normal” state, an        inhibitor can be added to demonstrate specificity. For example,        the TrkA inhibitor K252a or AG879 (available from Biomol        Research Laboratories, Inc.) can be used to demonstrate that the        agonist is working via the TrkA receptor. If the agonist is        specifically targeted to the TrkA receptor, it should not work        in the presence of a TrkA inhibitor.

EXAMPLE 3 Screening for G-Protein Coupled Receptor (GPCR) Agonists I.Background

The calcium ion is a very important messenger in cells. Theconcentration of free Ca²⁺ is extremely low (10⁻⁷ M) in the cytosolcompared to the extracellular fluid (10⁻³ M) or to the endoplasmicreticulum. A variety of reactions such as receptor-ligand interactionsmediate changes in the concentration of free intracellular calcium([Ca²⁺] i). The change in intracellular calcium occurs rapidly.

II. Experimental Design

The Fluo-3 AM (Molecular Probes Cat. No. 1241) absorption spectrum iscompatible with excitation at 488 nm by argon-ion laser sources. UponCa²⁺ binding, there is a large increase in fluorescence intensity.Fluo-3 AM is cell permeant. A variety of cell types can be utilizedincluding, CHO-K1, HEK-293 and COS.

Assays are conducted as follows:

-   -   1. Cells (introduced via inlet A. a), and 2 μM Fluo-3 calcium        indicator (introduced via inlet A. b) are added to main flow        channel A, such that there is one cell per “chamber”.    -   2. Incubate cells and fluo-3 for 30 minutes in main flow        channel A. Conditions for incubations are 5% CO₂ and 37° C.    -   3. After 30 minutes, the cells are pumped to main flow channel B        and washed with an indicator-free medium in chambers along        channel B to remove any dye that is non-specifically associated        with the surface, and then incubated for an additional 30        minutes.    -   4. After 30 minutes, potential agonists are added via branch        flow channels 1, 2, 3, to the cells in main flow channel B.    -   5. Calcium mobilization is assessed at several time points        (e.g., 1 sec, 5 sec, and 30 sec, etc.). After time t,        fluorescent signals are detected using an air-cooled argon laser        with excitation at 488 nm and detection at 526 nm.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent or patent application were specifically andindividually indicated to be so incorporated by reference.

1. A microfluidic device for conducting assays, comprising: (a) a mainflow channel adapted to allow the flow of a solution therethrough; (b) aplurality of branch flow channels, wherein each branch flow channel isin fluid communication with the main flow channel and comprises adetection region; (c) a plurality of control channels; and (d) aplurality of valves operatively disposed with respect to the main flowchannel and/or the plurality of branch flow channels to regulate flow ofthe solution through the main and branch flow channels, wherein each ofthe valves comprises one of the control channels and an elastomericsegment that is deflectable into or retractable from the main or branchflow channel upon which the valve operates in response to an actuationforce applied to the control channel, the elastomeric segment whenpositioned in the flow channel restricting solution flow therethrough.2. The microfluidic device of claim 1, wherein the control channel ofeach valve is separated from the flow channel in which the valveoperates by the elastomeric segment.
 3. The microfluidic device of claim1 further comprising a solution inlet in fluid communication with themain flow channel for introduction of the solution.
 4. The microfluidicdevice of claim 3 further comprising a second solution inlet for eachbranch flow channel in fluid communication therewith for introduction ofa second solution.
 5. The microfluidic device of claim 3 furthercomprising an auxiliary inlet in fluid communication with the main flowchannel.
 6. The microfluidic device of claim 5 further comprising aninlet flow channel in fluid communication with the main flow channel,and wherein the solution inlet and the auxiliary inlet are in fluidcommunication with the inlet flow channel.
 7. The microfluidic device ofclaim 1 further comprising a detector operatively disposed with respectto at least one of the detection regions to detect an event or agentwithin the detection region.
 8. The microfluidic device of claim 7,wherein the detector detects an optical signal from the detectionregion.
 9. The microfluidic device of claim 8, wherein the detector candetect a fluorescence emission, fluorescence polarization orfluorescence resonance energy transfer.
 10. The microfluidic device ofclaim 9, wherein the detector can perform time-resolved fluorescencemeasurements of fluorescence correlation spectroscopy.
 11. Themicrofluidic device of claim 8, wherein the detector is an opticalmicroscope, a confocal microscope or a laser scanning confocalmicroscope.
 12. The microfluidic device of claim 7, wherein the detectoris a non-optical sensor selected from the group consisting of atemperature sensor, a conductivity sensor, a potentiometric sensor andan amperometric sensor.
 13. The microfluidic device of claim 1, whereineach branch flow channel is adapted to allow the flow of a secondsolution therethrough.
 14. The microfluidic device of claim 1, whereinthe main flow channel is one of a plurality of main flow channels, eachadapted to allow the flow of a first solution therethrough and in fluidcommunication with each of the branch flow channels.
 15. Themicrofluidic device of claim 1, wherein each of the branch flow channelsintersect with the main flow channel at a different intersection, andfurther comprising a chamber located at each of the intersections, thechamber being adapted to collect solution therein.
 16. The microfluidicdevice of claim 1 further comprising a plurality of pumps, and whereineach pump is operatively disposed with respect to one of the branch flowchannels such that solution flow through each of the branch flowchannels can be regulated by one of the pumps.
 17. The microfluidicdevice of claim 16, wherein each of the plurality of pumps comprises atleast three control channels each formed within an elastomeric materialand separated from the branch flow channel by a section of anelastomeric membrane, the membrane being deflectable into the branchflow channel in response to an actuation force.
 18. The microfluidicdevice of claim 1 further comprising a plurality of mixers, wherein eachmixer is operatively disposed with respect to one of the branch flowchannels and is adapted to receive and mix different solutions flowingthrough one of the branch flow channels.
 19. The microfluidic device ofclaim 18 further comprising a temperature controller operativelydisposed to regulate temperature within the mixer.
 20. The microfluidicdevice of claim 18, wherein the mixer comprises (i) an inlet section andan outlet section; (ii) a looped flow channel in fluid communicationwith the inlet and outlet section; and (iii) a pump operativelyconnected to the looped flow channel, wherein the pump comprises aplurality of control channels, each separated from the looped flowchannel by a segment of an elastomeric membrane that is deflectable intothe looped flow channel in response to an actuation force, and whereinthe mixer is in fluid communication with at least one of the flowchannels such that solution from the flow channel can enter via theinlet section and reenter the flow channel via the outlet section, andsolution within the mixer can be cycled through the looped flow channelby the pump.
 21. The microfluidic device of claim 20, wherein thedetection region comprises the mixer.
 22. The microfluidic device ofclaim 1 further comprising a temperature controller.
 23. Themicrofluidic device of claim 22 further comprising a plurality oftemperature controllers, wherein each temperature controller isoperatively disposed with respect to one of the branch channels.
 24. Themicrofluidic device of claim 22, wherein the temperature controller isdisposed to regulate temperature within the detection region.
 25. Themicrofluidic device of claim 1 further comprising a plurality ofseparation units, wherein each separation unit is operatively disposedwith respect to one of the branch flow channels.
 26. The microfluidicdevice of claim 25, wherein each separation unit comprises asemi-penneable membrane that separates one of the branch flow channelsand a collection channel, the membrane allowing passage of certainagents in a solution flowing through the branch flow channel to passthrough the semi-penneable membrane into the collection flow channel.27. The microfluidic device of claim 25, wherein the separation unitcomprises a separation material that separates molecules on the basis ofaffinity, size, charge or mobility.
 28. The microfluidic device of claim1 further comprising: (e) an inlet in fluid communication with the mainflow channel for introduction of the solution and an inlet for eachbranch flow channel in fluid communication therewith for introduction ofa second solution; (f) a plurality of chambers positioned at pointswhere the main flow channel and the branch flow channels intersect, thechambers being adapted to collect solution therein; (g) a plurality ofpumps, each pump being operatively disposed with respect to one of thebranch flow channels such that solution flow through each of the branchflow channels can be regulated by one of the pumps; and (h) a pluralityof mixers, each mixer being operatively disposed with respect to one ofthe branch flow channels and being adapted to receive and mix differentsolutions flowing through one of the branch flow channels.
 29. Themicrofluidic device of claim 1 further comprising a pair of storagevalves operatively disposed with respect to each of the branch flowchannels, the pair of storage valves being disposed with respect to oneanother such that when the elastomeric segment of each storage valve ofthe pair extends into the branch flow channel a holding space is formedbetween the segments in which the solution can be retained.
 30. Themicrofluidic device of claim 29 further comprising a plurality ofcontrol valves operatively disposed with respect to the main flowchannel, and wherein the control valves are positioned along the mainflow channel such that by selectively actuating the control valvessolution can be controllably introduced into the branch flow channels;and the control valves each comprise one of the control channels and anelastomeric segment that is deflectable into or retractable from themain flow channel in response to an actuation force applied to thecontrol channel.
 31. The microfluidic device of claim 30, wherein theelastomeric segments of at least one pair of storage valves eachcomprise one or more protrusions, the protrusions adapted to allow forthe solution to flow through the holding space once the elastomericsegments are deflected into the branch flow channel while capable ofretaining a particle that is present in the solution within the holdingspace of the at least one pair of storage valves.
 32. The microfluidicdevice of claim 30, wherein the branch flow channel is formed within anelastomeric material and a segment of the branch flow channel oppositethe elastomeric segment of each storage valve of at least one pair ofstorage valves comprises one or more elastomeric protrusions, theprotrusions adapted to allow for solution to flow through the holdingspace once the elastomeric segment is deflected into the branch flowchannel while retaining a particle that is present in the solutionwithin the holding space of the at least one pair of valves.
 33. Themicrofluidic device of claim 30, wherein the branch flow channels areformed within an elastomeric material; the elastomeric segments of atleast one pair of storage valves comprise one or more protrusions; asegment of the branch flow channel opposite the elastomeric segments ofthe at least one storage valve pair comprises one or more protrusions,and wherein the protrusions of the elastomeric segment and theprotrusions of the branch flow channel are adapted to allow for solutionto flow through the holding space of the at least one valve pair oncethe elastomeric segments are deflected into the branch flow channelwhile retaining a particle that is present in the solution within theholding space.
 34. The microfluidic device of claim 29 furthercomprising a plurality of chambers positioned at points where an inletflow channel and the branch flow channels intersect, the chambersadapted for storing solution.
 35. The microfluidic device of claim 29further comprising a plurality of branch control valves, wherein abranch control valve is operatively disposed with respect to each of thebranch flow channels and each branch control valve comprises anelastomeric segment that separates one of the branch flow channels andone of the control channels and that is deflectable into or retractablefrom the branch flow channel upon which the valve operates in responseto an actuation force applied to the control channel.
 36. Themicrofluidic device of claim 29, wherein each branch flow channel is incommunication with a pump, the pump comprising at least three of thecontrol channels, each formed within an elastomeric material andseparated from one of the branch flow channels by an elastomericmembrane, the membrane being deflectable into the branch flow channel inresponse to an actuation force.
 37. The microfluidic device of claim 29,wherein each branch flow channel comprises a particle enrichment sectionthat selectively retains particles of interest.
 38. The microfluidicdevice of claim 37, wherein the enrichment section contains a ligandthat is capable of specifically binding to particle (s) that are presentin the solution.
 39. The microfluidic device of claim 38, wherein thedetection region includes the holding space and a detector is disposedto detect particle (s) within the holding spaces within the plurality ofbranch channels.
 40. The microfluidic device of claim 29, wherein thedetection region includes the hold space.
 41. The microfluidic device ofclaim 29, wherein the detection region is located at a section of thebranch flow channel other than the holding space.
 42. A microfluidicdevice for conducting cellular assays, comprising: (a) a main flowchannel; (b) an input adapted to receive a first solution and in fluidcommunication with the main flow channel, whereby solution introducedinto the input can flow into the main flow channel; (d) a plurality ofcontrol channels; (e) a plurality of chambers positioned along the mainflow channel and in fluid communication therewith, such that solutionflowing through the main flow channel can be stored in the chambers; (f)a plurality of branch flow channels, wherein each branch flow channel isin fluid communication with a branch inlet, each branch inlet adapted toreceive a second solution and in fluid communication with one of thechambers, different branch flow channels being in fluid communicationwith different chambers; (g) a plurality of storage valves, wherein apair of storage valves are operatively disposed with respect to each ofthe branch flow channels, and wherein each of the storage valvescomprises an elastomeric segment that separates one of the branch flowchannels and one of the control channels and that is deflectable into orretractable from the flow channel upon which the valve operates inresponse to an actuation force applied to the control channel, thestorage valves of a pair being disposed with respect to one another suchthat when the elastomeric segment of each storage valve of the pairextends into the branch flow channel a holding space is formed betweenthe segments in which one or more cells can be retained; and (h) one ormore pumps to transport the first and/or second solution through thebranch flow channels.
 43. The microfluidic device of claim 42, whereineach pump comprises at least three control channels, each formed withinan elastomeric material and separated from one of the branch flowchannels by a section of an elastomeric membrane, the membrane beingdeflectable into the branch flow channel in response to an actuationforce.
 44. A method of conducting a cellular analysis, comprising (a)providing a microfluidic device comprising (i) a flow channel comprisinga detection region; (ii) a plurality of control channels; and (iii) apair of storage valves operatively disposed with respect to the flowchannel, wherein each storage valve comprises one of the controlchannels, which control channel is separated from the flow channel by anelastomeric membrane, the elastomeric membrane being deflectable intothe flow channel in response to an actuation force applied to thecontrol channel, the two control channels of the storage valves beingdisposed relative to one another such that deflection of the elastomericmembrane of the respective control channels into the flow channel formsa holding space within the flow channel; (b) introducing a samplecontaining one or more cells into the flow channel; (c) actuating thestorage valves to hold at least one of the cells within the holdingspace; and (d) performing an assay by contacting the cells within theholding space with a solution containing one or more assay agents.